Quinoline amides and methods of using same

ABSTRACT

The present invention provides novel compounds that are quinoline foldamers. Such foldamers stabilize and bind to islet amyloid polypeptide (IAPP). In certain embodiments, the quinoline foldamers of the invention are soluble and cross biological membranes without cellular assistance. The present invention further provides novel method of preventing or treating diabetes in a subject in need thereof by administering to the subject an effective amount of at least one quinoline foldamer of the invention. The present invention further provides novel method of preventing or treating a neurodegenerative disease caused by a misfolded and/or unstructured protein in a subject in need thereof by administering to the subject an effective amount of at least one quinoline foldamer of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of, and claims priority to, U.S.Application No. 15,769,914, filed April, 2018, now issued as U.S. Pat.No. 11,135,213, which is a 35 U.S.C. § 371 national phase applicationfrom, and claims priority to, International Application No.PCT/US2016/059007, filed Oct. 27, 2016, and published under PCT Article21(2) in English, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/247,572, filed Oct. 28, 2015, which ishereby incorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM094693 awardedby National Institutes of Health. The government has certain rights inthe invention.

SEQUENCE LISTING

The ASCII text filed name “047162-7069US2_Sequence_Listing_ST25” createdon Aug. 26, 2021, comprising 0.876 Kbytes, is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Biopolymers are distinguished from artificial polymers by the presenceof a specified sequence of precursors, which allows them to fold to aspecific structure. A tiny set of precursors (four for DNA/RNA, andtwenty for proteins) creates a breadth of folds and functions essentialto life. In contrast, synthetic polymers have access to an essentiallyunlimited array of precursor variants; however, the lack of sequencecontrol and unique conformation results in a breadth of function that isdwarfed by biology.

Synthetic foldamers seek to join the best of these two worlds. Newscaffolds with specifiable sequences permit folded and functionalstructures to be successfully designed. For instance, foldamers based onoligomers of arylamides, β-peptide, α/β-peptide and peptoids have beendesigned to be an antimicrobial agent, agonist of GLP-1 receptor, aninhibitor of HIV-cell fusion and an antagonist of VEGF receptor 2,respectively. The achievable specificities of these small moleculesrivals biopolymers.

Dynamic binding modes, such as conformational-selection, are oftenobserved in protein-ligand interactions, such as for the anti-cancerdrug Gleevec. Gleevec binds to structurally identical sites in Src andits homologue, Abl. Nevertheless, there is a >1,000-fold difference inbinding affinity that can be explained by differences in proteindynamics. It stands to reason that the internal degrees of freedom ofligands should be similarly exploitable. However, ligand dynamics areviewed unfavourably as a result of entropy loss upon binding. Thenon-covalent and reversible stabilization of foldamer structure presentsan opportunity to limit entropic loss upon protein binding withoutsacrificing opportunities and potential benefits of dynamic binding.

There is a need in the art for novel foldamers, which can be used totreat diseases in a mammal. The present invention addresses this unmetneed.

BRIEF SUMMARY OF THE INVENTION

The invention provides a compound. The invention further provides apharmaceutical composition comprising at least one compound of theinvention and at least one pharmaceutically acceptable carrier. Theinvention further provides a method of preventing or treating diabetesin a subject in need thereof. The invention further provides a method ofpreventing or treating a neurodegenerative disease caused by a misfoldedor unstructured protein. The invention further provides a method ofincreasing the cell membrane permeability of a molecule CARGO, whereinCARGO is selected from the group consisting of an oligonucleotide,oligodeoxynucleotide, small molecule and polypeptide.

In certain embodiments, the compound is the compound of formula (I), ora salt, solvate, or N-oxide thereof, and any combinations thereof:

wherein each occurrence of R¹ is independently selected from the groupconsisting of —OH, —O(C₁-C₆)alkyl, —O(C₁-C₆)haloalkyl,—O(C₁-C₆)heteroalkyl, —O(CH₂)_(m)C(═O)OR⁵, —OC(═O)R⁵, —NH₂, —SH, —SO₃Hand —PO(OH)₂; wherein R² is selected from the group consisting of—(C₁-C₆)alkyl, —(C₁-C₆)heteroalkyl, —OR⁵, —(C₃-C₁₀)heterocyclyl, aryland heteroaryl, wherein the alkyl, hetereoalkyl, heterocyclyl, aryl orheteroaryl group is optionally substituted; wherein R³ and R⁴ areindependently selected from the group consisting of H,—(C═O)₀₋₁(C₁-C₆)alkyl, —(C═O)₀₋₁(C₃-C₅)cycloalkyl,—(C═O)₀₋₁(C₁-C₆)heteroalkyl, —(C═O)₀₋₁aryl, and —(C═O)₀₋₁heteroaryl,wherein the alkyl, cycloalkyl, aryl or heteroaryl group is optionallysubstituted; or R³ and R⁴, together with the nitrogen to which R³ and R⁴are connected, form —(C₃-C₁₀)heterocyclyl or —NO₂; wherein eachoccurrence of R⁵ is independently selected from the group consisting ofH, —(C₁-C₆)alkyl, —(C₁-C₆)heteroalkyl, —(C₃-C₅)cycloalkyl,—(C₄-C₁₀)heterocyclyl, aryl, and —(C₅-C₁₀)heteroaryl, wherein the alkyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group isoptionally substituted; wherein each occurrence of m is independently aninteger ranging from 1 to 5; wherein k is an integer ranging from 1 to5; and wherein the compound of formula (I) has a net negative charge atphysiological pH.In certain embodiments, the compound is the compound of formula (II), ora salt, solvate, or N-oxide thereof, and any combinations thereof:

wherein each occurrence of R¹ is independently selected from the groupconsisting of —OH, —O(C₁-C₆)alkyl, —O(C₁-C₆)haloalkyl,—O(C₁-C₆)heteroalkyl, —O(CH₂)mC(═O)OR⁵, —OC(═O)R⁵, —NH₂, —SH, —SO₃H and—PO(OH)₂; wherein (i) R² is -X1-LINKER-CARGO or (ii) R³ is-X2-LINKER-CARGO; wherein X1 is a bond, —NH—, —O— or —S—; wherein X2 is—C(═O)— or —C(═S)—; wherein LINKER comprises a group consisting of—(CH₂)_(n)—, —(CH₂CH₂O)_(n)— and -(AA)_(n)-, wherein each occurrence ofn is independently an integer ranging from 1 to 10 and wherein eachoccurrence of AA is independently a naturally occurring amino acid,further wherein -X-LINKER optionally further comprises a disulfide bond;and wherein CARGO is a molecule selected from an oligonucleotide,oligodeoxynucleotide, small molecule and polypeptide; wherein if R² is-X1-LINKER-CARGO, then R³ and R⁴ are independently selected from thegroup consisting of H, —(C═O)₀₋₁(C₁-C₆)alkyl,—(C═O)₀₋₁(C₃-C₅)cycloalkyl, —(C═O)₀₋₁(C₁-C₆)heteroalkyl, —(C═O)₀₋₁aryl,and —(C═O)₀₋₁heteroaryl, wherein the alkyl, cycloalkyl, aryl orheteroaryl group is optionally substituted; or R³ and R⁴, together withthe nitrogen to which R³ and R⁴ are connected, form—(C₃-C₁₀)heterocyclyl or —NO₂; and wherein if R³ is -X2-LINKER-CARGO,then R² is selected from the group consisting of —(C₁-C₆)alkyl,—(C₁-C₆)heteroalkyl, —OR⁵, —(C₃-C₁₀)heterocyclyl, aryl, and heteroaryl,wherein the alkyl, heteroalkyl, heterocyclyl, aryl or heteroaryl groupis optionally substituted; and R⁴ is H; wherein each occurrence of R⁵ isindependently selected from the group consisting of H, —(C₁-C₆)alkyl,—(C₁-C₆)heteroalkyl, —(C₃-C₅)cycloalkyl, —(C₄-C₁₀)heterocyclyl, aryl,and —(C₅-C₁₀)heteroaryl, wherein the alkyl, heteroalkyl, cycloalkyl,heterocyclyl, aryl, or heteroaryl group is optionally substituted;wherein each occurrence of m is independently an integer ranging from 1to 5; wherein k is an integer ranging from 1 to 5; and wherein thecompound of formula (II) has a net negative charge at physiological pH.

In certain embodiments, at least one occurrence of R¹ is selected fromthe group consisting of —O(CH₂)_(m)C(═O)OH, —SO₃H and —PO(OH)₂. In otherembodiments, at least one occurrence of R¹ is —O(CH₂)_(m)COOH. In yetother embodiments, at least two occurrences of R¹ are independentlyselected from the group consisting of —O(CH₂)_(m)C(═O)OH, —SO₃H and—PO(OH)₂. In yet other embodiments, at least two occurrences of R¹ areindependently —O(CH₂)_(m)C(═O)OH.

In certain embodiments, every other quinoline group in (I) has a R¹independently selected from the group consisting of —O(CH₂)_(m)C(═O)OH,—SO₃H and —PO(OH)₂ at the 4-position of the respective ring.

In certain embodiments, if a given quinoline group in (I) has a R¹selected from the group consisting of —O(CH₂)_(m)C(═O)OH, —SO₃H and—PO(OH)₂ at the 4-position of the ring, the quinoline groups to whichthe given quinoline group is covalently linked have R¹ substituents thatare independently selected from the group consisting of —OH,—O(C₁-C₆)alkyl, —O(C₁-C₆)haloalkyl, —O(C₁-C₆)heteroalkyl, —OC(═O)R⁵,—NH₂, and —SH, at the 4-position of the corresponding rings, wherein R⁵is not H.

In certain embodiments, k is 3. In other embodiments, each occurrence ofR¹ is independently selected from the group consisting of —OCH₂CH₃ and—OCH₂COOH; NR³R⁴ is —NO₂; R² is —OMe; and k is 3.

In certain embodiments, any two quinoline groups that are covalentlylinked do not have the same R¹ substituent.

In certain embodiments, the compound is the compound of formula (III) or(IV):

In certain embodiments, at physiological pH the compound of formula (I)has a net negative charge that is selected from the group consisting of−1, −2, −3, −4, −5, and −6.

In certain embodiments, CARGO comprises a detectable label. In otherembodiments, at physiological pH the compound of formula (II) in theabsence of CARGO has a net negative charge that is selected from thegroup consisting of −1, −2, −3, −4, −5, and −6.

In certain embodiments, the compound is water soluble. In otherembodiments, the compound is capable of passively penetrating a cellmembrane.

In certain embodiments, the compound of formula (II) has higher cellmembrane permeability than CARGO.

In certain embodiments, X1-LINKER or X2-LINKER is cleaved within a cell.

In certain embodiments, the compound is further coupled with adetectable label. In other embodiments, the detectable label is selectedfrom the group consisting of a radioisotope, stable isotope,fluorophore, electron dense metals, biotin, DNA, RNA, antibody epitope,spin label, reactive peptide tag, quantum dot and any combinationsthereof.

In certain embodiments, the pharmaceutical composition further comprisesat least one additional therapeutic agent.

In certain embodiments, the method comprises administering to thesubject a therapeutically effective amount of at least one compound ofthe invention. In other embodiments, the method further comprisesadministering to the subject at least one additional therapeutic agentthat treats or prevents diabetes. In yet other embodiments, the methodfurther comprises administering to the subject at least one additionaltherapeutic agent that treats or prevents the neurodegenerative disease.

In certain embodiments, the compound and the at least one additionaltherapeutic agent are co-administered to the subject. In otherembodiments, the compound and the at least one additional therapeuticagent are co-formulated.

In certain embodiments, the diabetes is type I or type II diabetes. Inother embodiments, the protein comprises α-synuclein, tau or Aβ. In yetother embodiments, the neurodegenerative disease comprises Parkinson'sor Alzheimer's Disease. In yet other embodiments, the subject is ahuman.

In certain embodiments, the method comprises derivatizing CARGO to forma compound of formula (II). In other embodiments, the compound iscleaved within the cell releasing CARGO or a derivative thereof, whereinthe derivative of CARGO has essentially the same biological activity asCARGO.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of specific embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings specific embodiments. It should be understood,however, that the invention is not limited to the precise arrangementsand instrumentalities of the embodiments shown in the drawings.

FIG. 1A depicts chemical structure of ADM 116, also denoted as compoundof formula (III). Arrows indicate surface exposed functional moieties.FIG. 1B depicts three-dimensional stick (top) and sphere (bottom) modelof ADM 116. Arrows indicate surface exposed functional moieties. FIG. 1Cillustrates the primary sequence of human islet amyloid polypeptide(IAPP; SEQ ID NO:1), which is C-terminally amidated. FIG. 1D depictsthree-dimensional stick model of pentaquinoline, ADM-1161. Arrowsindicate surface exposed functional moieties.

FIG. 2 comprises a series of images illustrating time dependent uptakeof IAPP and toxicity. At time zero, 100 nM IAPPA594 with (toxic) andwithout (non-toxic) an additional 13 μM of unlabelled IAPP was added toculture media of INS-1 cells. Confocal images were taken at theindicated time points. Scale bar=10 μm. Graphic inset: Colourimetricevaluation of toxicity (CTB) and apoptosis (Caspase) at the toxiccondition relative to vehicle-only controls.

FIGS. 3A-3B illustrate effects of ligands on the kinetics of IAPP fibreformation. FIG. 3A illustrates 10 μM IAPP fibre formation over timecatalysed by unilamellar vesicles (630 μM monomer units,dioleoylphosphatidylglycerol (DOPB):dioleoylphosphatidylcholine(DOPC)=1:1, 100 nm). FIG. 3B illustrates inhibition with the indicatedsmall molecules performed at IAPP:small molecule=1:1 (reactionmidpoints, t₅₀). Inset illustrates statistics of dose dependence ofinhibition of fibre formation by ADM 116.

FIGS. 4A-4C illustrate rescue of INS-1 cells from IAPP induced toxicity.FIG. 4A illustrates statistics of toxic effect of 13 μM IAPP applied attime-zero to INS-1 cells and measured 48 h later using Cell-Titer Blue(CTB). Data are shown for IAPP alone and with equimolar ratio of theindicated small molecule co-added at 1:1 ratio. Data are renormalized tothe toxicity induced only by IAPP. FIG. 4B illustrates dose dependenceof toxic rescue by ADM-3 and ADM-116. FIG. 4C illustrates relativetoxicity of 13 μM IAPP in which a time delay is introduced betweenaddition of IAPP and addition of molecule. Error bars in FIGS. 4A-4C arestandard deviations from three sets of experiments conducted on separateoccasions.

FIGS. 5A-5D illustrate small molecules binding to IAPP and theirstructural effects. FIG. 5A illustrates relative binding of ADM-116 toIAPP using 25 nM ADM-116F, obtained by measuring diffusion byfluorescence correlation spectroscopy (FCS) as a function of IAPP. Insetillustrates representative raw autocorrelation data (black) and twocomponent fit (red) for 0 nM and 250 nM IAPP respectively. FIG. 5Billustrates association constants for the indicated small molecule withhuman IAPP measured by Isothermal titration calorimetry (ITC).ND=undetectable binding. Inset illustrates representative raw data andfit for ADM-116 binding plotted as a function of stoichiometry. FIG. 5Cillustrates free energy and entropy contributions to binding by ITC forthe indicated compounds. FIG. 5D illustrates far UV CD spectra of 25 μMIAPP in the presence of large unilamellar vesicles (LUVs) (1.2 mM inmonomeric units, DOPG:DOPC=1:1, 100 nm). Shown are two time points forIAPP alone (blue, black) and with addition of stoichiometric ADM-116 att=0. ADM-116 without IAPP is shown as control (orange).

FIGS. 6A-6C illustrate membrane translocation and partitioning. FIG. 6Adepicts confocal fluorescence images of INS 1 cells incubated for 12 hwith 200 nM ADM-116_(F) or 200 nM ADM-3F. Conditions shown are with andwithout addition of a further 15 μM of unlabelled small molecule. FIG.6B depicts confocal fluorescence images giant plasma membrane derivedvesicles (GPMVs) prepared from INS-1 cells incubated for 24 h with 200nM ADM-11⁶F or ADM-3_(F) mixed with 10 μM of unlabelled compound. FIG.6C illustrates calculated versus measured octanol:water partitioncoefficients for the three molecules: ADM-116, ADM-116▮, and ADM-3.

FIGS. 7A-7D illustrate colocalization of IAPP and ADM-116. FIG. 7Acomprises a series of images illustrating INS-1 cells exposed to arescued condition doped with fluorescent variants of protein and ligand(13 μM IAPP, 15 μM ADM-116, 100 nM IAPP_(A594) and 200 nM ADM-116_(F)).The small molecule and IAPP were co-introduced to the culture media.FIG. 7B comprises a series of images illustrating INS-1 cells exposed toa rescued condition doped with fluorescent variants of protein andligand (13 μM IAPP, 15 μM ADM-116, 100 nM IAPP_(A594) and 200 nMADM-116_(F)). The small molecule was added 20 hours after IAPPintroduced to the culture media. FIG. 7C comprises a series of imagesillustrating INS-1 cells exposed to a rescued condition doped withfluorescent variants of protein and ligand (13 μM IAPP, 15 μM ADM-116,100 nM IAPP_(A594) and 200 nM ADM-116_(F)). The rescued condition wasthe same as that depicted in FIG. 7B, but without the unlabelledcomponents and initial incubation with IAPP preformed for 18 h. Confocalfluorescence images were collected at the acceptor's emission wavelengthusing donor (left) or acceptor excitation light (middle). FRET wascomputed from these channels and are shown as white dots on a DIC imageof a representative cell. Only areas showing a FRET efficiency of >0.4are shown as values below this are indistinguishable from background.Background FRET was determined using parallel experiments which includeda further 13 μM of unlabelled IAPP (FIG. 14). FIG. 7D is a FREThistogram depicting the total counts at the indicated FRET efficienciesacross ˜50 regions of interest.

FIGS. 8A-8C illustrate selection, perturbation and activity of ADM-116analogs. FIG. 8A depicts visible CD of 25 μM ADM-116 or ADM-1161 in thepresence of 25 μM IAPP. FIG. 8B depicts visible CD of ADM-116 and eachof two chiral variants designed to introduce bias into the ADM-116enantiomer distribution. FIG. 8C depicts comparison of the fibreformation inhibition and toxic rescue activities of enantiomer biasedconstructs of ADM-116. Experiment conditions match those used in FIGS.3A-3B and FIGS. 4A-4C respectively.

FIGS. 9A-9B illustrate kinetic profiles of IAPP self-assembly. FIG. 9Aillustrates three representative kinetic profiles for a standardreaction of 10 μM IAPP catalysed by the presence of 630 μM LUVs(DOPG:DOPC=1:1, 100 nm). Inset illustrates a representative sigmoid fit(magenta) used to extract reaction midpoints, t₅₀. FIG. 9B illustratesrepresentative comparison of the kinetic profiles of (10 μM) IAPPfibrillation in the absence (black) and presence (red) of 630 μM LUVs.All experiments were conducted at least in triplicate with errorsreported as ±one standard deviation.

FIGS. 10A-10B illustrate images of ADM-116 inhibited assembly. FIG. 10Adepicts negative stain transmission electron microscopy (TEM) images ofliposome catalysed IAPP (10 μM) self-assembly in the absence of 10 μMADM-116. FIG. 10B depicts negative stain TEM images of liposomecatalysed IAPP (10 μM) self-assembly in the presence of 10 μM ADM-116.

FIG. 11 illustrates intrinsic toxicity of selected small molecules.INS-1 cells were incubated with 13 μM of each of the indicated smallmolecules. Viability was assayed after 48 h using Cell-Titer Blue (CTB)and compared to carrier-only controls. Error bars represent the standarddeviation in the mean of four replicates.

FIG. 12 illustrates effects of ADM-116 on the assembly kinetics ofIAPP₂₀₋₂₉. Representative kinetic profile of amyloid assembly by 200 μMof the 10-residue peptide, IAPP₂₀₋₂₉. Reactions are shown in the absenceand presence of 200 μM ADM-116 at equimolar ratio.

FIGS. 13A-13B illustrate colocalization of IAPP and ADM-3. INS-1 cellswere exposed to a rescued condition (13 μM IAPP and 15 μM ADM-3) dopedwith 200 nM ADM-3F and 100 nM IAPP_(A594)). FIG. 13A comprises a seriesof images of INS-1 cells when IAPP and small molecule were added at thesame time to culture media. FIG. 13B comprises a series of images ofINS-1 cells when ADM-3 was added 20 h after addition of IAPP.

FIG. 14 illustrates determination of background Forster resonance energytransfer (FRET) in confocal imaging. Confocal imaging and processing forFRET was conducted in a manner matched to that used in the FIGS. 7A-7D.Preparation of the cells was also matched, except that the 100 nMIAPP₅₉₄ was augmented with 13 μM unlabelled IAPP to dilute out anypossibility of structure-specific FRET taking place.

FIG. 15 illustrates the chemical structures of the small molecules:ADM116, ADM-116▮, ADM-▮116, ADM-116_(P), ADM-116_(M), ADM-3_(F), ADM-3,and ADM-116_(F).

FIGS. 16A-16B illustrate the conformational changes induced by ADM-116.FIG. 16A illustrates visible CD spectra of 25 μM ADM-116 or ADM-116P inthe presence of liposome (630 μM in monomer units, DOPG:DOPC=1:1, 100nm) and 25 μM IAPP. FIG. 16B illustrates far-UV CD spectra of 25 μM IAPPin the presence of ADM-116 or ADM-1161 at a ratio of 1:0.5 (IAPP:smallmolecule).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in part to the unexpected discovery ofnovel quinoline foldamers that bind to islet amyloid polypeptide (IAPP)and prevent the formation of (toxic) amyloid states of IAPP in a mammal.In certain embodiments, the compounds of the invention can be used totreat or prevent Type I and/or Type II diabetes in a mammal, such as ahuman. In other embodiments, the compounds of the invention can be usedto treat neurodegenerative diseases based on misfolded and/orunstructured proteins. Exemplary proteins contemplated within theinvention include α-synuclein (Parkinson's Disease), tau (Alzheimer'sDisease) and/or Aβ (Alzheimer's Disease).

The present invention further provides quinoline foldamer conjugates ofvarious biologically active compounds, wherein the conjugates haveimproved cell membrane permeability over the corresponding compounds.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, specific methods andmaterials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “abnormal,” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics that arenormal or expected for one cell or tissue type might be abnormal for adifferent cell or tissue type.

As used herein, the term “ADM-118” refers to the following compound, ora salt,

solvate or N-oxide thereof:

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

As used herein, the term “composition” or “pharmaceutical composition”refers to a mixture of at least one compound useful within the inventionwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition facilitates administration of the compound to a patient orsubject. Multiple techniques of administering a compound exist in theart including, but not limited to, intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. An appropriate therapeutic amount inany individual case may be determined by one of ordinary skill in theart using routine experimentation.

As used herein, the term “efficacy” refers to the maximal effect(E_(max)) achieved within an assay.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compounds prepared from pharmaceuticallyacceptable non-toxic acids, including inorganic acids, organic acids,solvates, hydrates, or clathrates thereof. Examples of such inorganicacids are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric,phosphoric, acetic, hexafluorophosphoric, citric, gluconic, benzoic,propionic, butyric, sulfosalicylic, maleic, lauric, malic, fumaric,succinic, tartaric, amsonic, pamoic, p-tolunenesulfonic, and mesylic.Appropriate organic acids may be selected, for example, from aliphatic,aromatic, carboxylic and sulfonic classes of organic acids, examples ofwhich are formic, acetic, propionic, succinic, camphorsulfonic, citric,fumaric, gluconic, isethionic, lactic, malic, mucic, tartaric,para-toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic,benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic(besylate), stearic, sulfanilic, alginic, galacturonic, and the like.Furthermore, pharmaceutically acceptable salts include, by way ofnon-limiting example, alkaline earth metal salts (e.g., calcium ormagnesium), alkali metal salts (e.g., sodium-dependent or potassium),and ammonium salts.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations.

As used herein, “pharmaceutically acceptable carrier” also includes anyand all coatings, antibacterial and antifungal agents, and absorptiondelaying agents, and the like that are compatible with the activity ofthe compound useful within the invention, and are physiologicallyacceptable to the patient. Supplementary active compounds may also beincorporated into the compositions. The “pharmaceutically acceptablecarrier” may further include a pharmaceutically acceptable salt of thecompound useful within the invention. Other additional ingredients thatmay be included in the pharmaceutical compositions used in the practiceof the invention are known in the art and described, for example inRemington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co.,1985, Easton, Pa.), which is incorporated herein by reference.

The terms “patient,” “subject,” or “individual” are used interchangeablyherein, and refer to any animal, or cells thereof whether in vitro or insitu, amenable to the methods described herein. In a non-limitingembodiment, the patient, subject or individual is a human.

As used herein, the term “potency” refers to the dose needed to producehalf the maximal response (EDSo).

As used herein, the term “small molecule” refers to a molecule of <2,000amu.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., a compoundof the invention (alone or in combination with another pharmaceuticalagent), to a patient, or application or administration of a therapeuticagent to an isolated tissue or cell line from a patient (e.g., fordiagnosis or ex vivo applications), who has a condition contemplatedherein, a symptom of a condition contemplated herein or the potential todevelop a condition contemplated herein, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect acondition contemplated herein, the symptoms of a condition contemplatedherein or the potential to develop a condition contemplated herein. Suchtreatments may be specifically tailored or modified, based on knowledgeobtained from the field of pharmacogenomics.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e. C₁₋₆means one to six carbon atoms) and including straight, branched chain,or cyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl. Most preferred is (C₁-C₆)alkyl, particularly ethyl,methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term “substituted alkyl” means alkyl as definedabove, substituted by one, two or three substituents selected from thegroup consisting of halogen, —OH, alkoxy, —NH₂, —N(CH₃)₂, —C(═O)OH,trifluoromethyl, —C—N, —C(═O)O(C₁-C₄)alkyl, —C(═O)NH₂, —SO₂NH₂,—C(═NH)NH₂, and —NO₂, preferably containing one or two substituentsselected from halogen, —OH, alkoxy, —NH₂, trifluoromethyl, —N(CH₃)₂, and—C(═O)OH, more preferably selected from halogen, alkoxy and —OH.Examples of substituted alkyls include, but are not limited to,2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

As used herein, the term “haloalkyl” means alkyl as defined above,substituted by one, two or three substituents selected from the groupconsisting of F, Cl, Br, and I.

As used herein, the term “heteroalkyl” by itself or in combination withanother term means, unless otherwise stated, a stable straight orbranched chain alkyl group consisting of the stated number of carbonatoms and one or two heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may be optionallyoxidized and the nitrogen heteroatom may be optionally quaternized orsubstituted. The heteroatom(s) may be placed at any position of theheteroalkyl group, including between the rest of the heteroalkyl groupand the fragment to which it is attached, as well as attached to themost distal carbon atom in the heteroalkyl group. Examples include:—O—CH₂—CH₂—CH₃, —CH₂—CH₂—CH₂—OH, —CH₂—CH₂—NH—CH₃, —CH₂—S—CH₂—CH₃,—NH—(CH₂)_(m)—OH (m=1-6), —N(CH₃)—(CH₂)_(m)—OH (m=1-6),—NH—(CH₂)_(m)—OCH₃ (m=1-6), and —CH₂CH₂—S(═O)—CH₃. Up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃, or—CH₂—CH₂—S—S—CH₃

As used herein, the term “alkoxy” employed alone or in combination withother terms means, unless otherwise stated, an alkyl group having thedesignated number of carbon atoms, as defined above, connected to therest of the molecule via an oxygen atom, such as, for example, methoxy,ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs andisomers. Preferred are (C₁-C₃) alkoxy, particularly ethoxy and methoxy.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine,more preferably, fluorine or chlorine.

As used herein, the term “cycloalkyl” refers to a mono cyclic orpolycyclic non-aromatic radical, wherein each of the atoms forming thering (i.e. skeletal atoms) is a carbon atom. In certain embodiments, thecycloalkyl group is saturated or partially unsaturated. In otherembodiments, the cycloalkyl group is fused with an aromatic ring.Cycloalkyl groups include groups having from 3 to 10 ring atoms.Illustrative examples of cycloalkyl groups include, but are not limitedto, the following moieties:

Monocyclic cycloalkyls include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.Dicyclic cycloalkyls include, but are not limited to,tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycycliccycloalkyls include adamantine and norbornane. The term cycloalkylincludes “unsaturated nonaromatic carbocyclyl” or “nonaromaticunsaturated carbocyclyl” groups, both of which refer to a nonaromaticcarbocycle as defined herein, which contains at least one carbon carbondouble bond or one carbon carbon triple bond.

As used herein, the term “heterocycloalkyl” or “heterocyclyl” refers toa heteroalicyclic group containing one to four ring heteroatoms eachselected from O, S and N. In certain embodiments, each heterocycloalkylgroup has from 4 to 10 atoms in its ring system, with the proviso thatthe ring of said group does not contain two adjacent O or S atoms. Inother embodiments, the heterocycloalkyl group is fused with an aromaticring. In certain embodiments, the nitrogen and sulfur heteroatoms may beoptionally oxidized, and the nitrogen atom may be optionallyquaternized. The heterocyclic system may be attached, unless otherwisestated, at any heteroatom or carbon atom that affords a stablestructure. A heterocycle may be aromatic or non-aromatic in nature. Incertain embodiments, the heterocycle is a heteroaryl.

An example of a 3-membered heterocycloalkyl group includes, and is notlimited to, aziridine. Examples of 4-membered heterocycloalkyl groupsinclude, and are not limited to, azetidine and a beta lactam. Examplesof 5-membered heterocycloalkyl groups include, and are not limited to,pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-memberedheterocycloalkyl groups include, and are not limited to, piperidine,morpholine and piperazine. Other non-limiting examples ofheterocycloalkyl groups are:

Examples of non-aromatic heterocycles include monocyclic groups such asaziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin, and hexamethyleneoxide.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e. having (4n+2) delocalized 7L (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings),wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples of arylgroups include phenyl, anthracyl, and naphthyl. Preferred examples arephenyl and naphthyl, most preferred is phenyl.

As used herein, the term “aryl-(C₁-C₃)alkyl” means a functional groupwherein a one- to three-carbon alkylene chain is attached to an arylgroup, e.g., —CH₂CH₂-phenyl. Preferred is aryl-CH₂— and aryl-CH(CH₃)—.The term “substituted aryl-(C₁-C₃)alkyl” means an aryl-(C₁-C₃)alkylfunctional group in which the aryl group is substituted. Preferred issubstituted aryl(CH₂)—. Similarly, the term “heteroaryl-(C₁-C₃)alkyl”means a functional group wherein a one to three carbon alkylene chain isattached to a heteroaryl group, e.g., —CH₂CH₂-pyridyl. Preferred isheteroaryl-(CH₂)—. The term “substituted heteroaryl-(C₁-C₃)alkyl” meansa heteroaryl-(C₁-C₃)alkyl functional group in which the heteroaryl groupis substituted. Preferred is substituted heteroaryl-(CH₂)—.

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. A polycyclic heteroaryl mayinclude one or more rings that are partially saturated. Examples includethe following moieties:

Examples of heteroaryl groups also include pyridyl, pyrazinyl,pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl,furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl,oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles and heteroaryls include indolyl(particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl,tetrahydroquinolyl, isoquinolyl (particularly 1-and 5-isoquinolyl),1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2-and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl,1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl,benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl),2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl(particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl,benzimidazolyl (particularly 2-benzimidazolyl), benzotriazolyl,thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, andquinolizidinyl.

As used herein, the term “substituted” means that an atom or group ofatoms has replaced hydrogen as the substituent attached to anothergroup. The term “substituted” further refers to any level ofsubstitution, namely mono-, di-, tri-, tetra-, or penta-substitution,where such substitution is permitted. The substituents are independentlyselected, and substitution may be at any chemically accessible position.In certain embodiments, the substituents vary in number between one andfour. In other embodiments, the substituents vary in number between oneand three. In yet other embodiments, the substituents vary in numberbetween one and two.

As used herein, the term “optionally substituted” means that thereferenced group may be substituted or unsubstituted. In certainembodiments, the referenced group is optionally substituted with zerosubstituents, i.e., the referenced group is unsubstituted. In otherembodiments, the referenced group is optionally substituted with one ormore additional group(s) individually and independently selected fromgroups described herein.

In certain embodiments, the substituents are independently selected fromthe group consisting of oxo, halogen, —CN, —NH₂, —OH, —NH(CH₃),—N(CH₃)₂, alkyl (including straight chain, branched and/or unsaturatedalkyl), substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, fluoro alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted alkoxy,fluoroalkoxy, —S-alkyl, S(═O)₂alkyl, —C(═O)NH[substituted orunsubstituted alkyl, or substituted or unsubstituted phenyl], —C(═O)N[Hor alkyl]₂, —OC(═O)N[substituted or unsubstituted alkyl]₂,—NHC(═O)NH[substituted or unsubstituted alkyl, or substituted orunsubstituted phenyl], —NHC(═O)alkyl, —N[substituted or unsubstitutedalkyl]C(═O)[substituted or unsubstituted alkyl], —NHC(═O)[substituted orunsubstituted alkyl], —C(OH)[substituted or unsubstituted alkyl]₂, and—C(NH₂)[substituted or unsubstituted alkyl]₂. In other embodiments, byway of example, an optional substituent is selected from oxo, fluorine,chlorine, bromine, iodine, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂, —CH₃,—CH₂CH₃, —CH(CH₃)₂, —CF₃, —CH₂CF₃, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —OCF₃,—OCH₂CF₃, —S(═O)₂—CH₃, —C(═O)NH₂, —C(═O)—NHCH₃, —NHC(═O)NHCH₃,—C(═O)CH₃, and —C(═O)OH. In yet one embodiment, the substituents areindependently selected from the group consisting of C₁₋₆ alkyl, —OH,C₁₋₆ alkoxy, halo, amino, acetamido, oxo and nitro. In yet otherembodiments, the substituents are independently selected from the groupconsisting of C₁₋₆ alkyl, C₁₋₆ alkoxy, halo, acetamido, and nitro. Asused herein, where a substituent is an alkyl or alkoxy group, the carbonchain may be branched, straight or cyclic, with straight beingpreferred.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Description

The present invention relates in part to unexpected discovery of novelcompounds, such as quinolone foldamers, that bind to islet amyloidpolypeptide (IAPP) and prevent the formation of (toxic) amyloid statesof IAPP in a mammal. The present invention also relates to a method fortreating or preventing diabetes by administering to a mammal in needthereof a therapeutically effective amount of a quinoline foldamer ofthe invention. The present invention also relates to a method fortreating or preventing neurodegenerative diseases based on misfoldedand/or unstructured proteins, such as α-synuclein (Parkinson's), tau(Alzheimer's) and/or Aβ (Alzheimer's).

Disordered proteins, such as those central to Alzheimer's andParkinson's, are particularly intractable for structure-targetedtherapeutic design. The present results demonstrate the capacity of asynthetic foldamer to capture structure in a disease relevant peptide.Oligoquinoline amides have a defined fold with a solvent-excluded corethat is independent of its outwardly projected, derivitizable moieties.IAPP is a 37-residue peptide co-packaged with insulin in pancreaticβ-cells (FIG. 1C). Aggregation of this peptide into amyloid fibres isobserved in type II diabetes, and pre-amyloid states of IAPP are toxinsresulting in f-cell death. The compounds of the invention comprisetetraquinolines that stabilize a pre-amyloid, α-helical conformation ofIAPP. The charged, polyanionic compounds of the invention are highlysoluble, yet cross biological membranes without cellular assistance.Without wishing to be limited by any theory, this may take place becausethe compounds are able to reversibly fold into a membrane-permeablestructure. The compounds of the invention antagonize toxicity long aftercellular uptake of IAPP is complete. These gains-of-function aredependent on the capacity of the foldamer to not only recognize IAPP,but also to transiently sample unfolded states. The compounds of theinvention dock specifically with intracellular IAPP and rescues f-cellsfrom apoptosis. Without wishing to be limited by any theory, the presentresults indicate that stabilizing non-toxic conformers of a plasticprotein can be a viable strategy for cytotoxic rescue.

Without wishing to be limited by any theory, the modes of action of IAPPare similar to those of polypeptides involved in degenerative diseases,including A3 peptide from Alzheimer's Disease and α-synuclein fromParkinson's disease. In all of these diseases, polypeptides arepredominantly disordered in aqueous solution, and undergo disorder toα-helical transitions upon interaction with biological membranes. Theseinteractions are associated with cell toxicity relevant to disease. Incertain embodiments, the compounds of the invention are useful intreating and/or preventing degenerative diseases including Alzheimer'sDisease and Parkinson's disease

Compounds

The compounds of the present invention may be synthesized usingtechniques well-known in the art of organic synthesis. The startingmaterials and intermediates required for the synthesis may be obtainedfrom commercial sources or synthesized according to methods known tothose skilled in the art.

In one aspect, the invention provides a compound of formula (I), a salt,solvate, or N-oxide thereof:

wherein each occurrence of R¹ is independently selected from the groupconsisting of —OH, —O(C₁-C₆)alkyl, —O(C₁-C₆)haloalkyl,—O(C₁-C₆)heteroalkyl, —O(CH₂)_(m)C(═O)OR⁵, —OC(═O)R⁵, —NH₂, —SH, —SO₃Hand —PO(OH)₂;

wherein R² is selected from the group consisting of —(C₁-C₆)alkyl,—(C₁-C₆)heteroalkyl, —OR⁵, —(C₃-C₁₀)heterocyclyl, aryl and heteroaryl,wherein the alkyl, hetereoalkyl, heterocyclyl, aryl or heteroaryl groupis optionally substituted;

wherein R³ and R⁴ are independently selected from the group consistingof H, —(C═O)₀₋₁(C₁-C₆)alkyl, —(C═O)₀₋₁(C₃-C₅)cycloalkyl,—(C═O)₀₋₁(C₁-C₆)heteroalkyl, —(C═O)₀₋₁aryl, and —(C═O)₀₋₁heteroaryl,wherein the alkyl, cycloalkyl, aryl or heteroaryl group is optionallysubstituted; or R³ and R⁴, together with the nitrogen to which R³ and R⁴are connected, form —(C₃-C₁₀)heterocyclyl or —NO₂;

wherein each occurrence of R⁵ is independently selected from the groupconsisting of H, —(C₁-C₆)alkyl, —(C₁-C₆)heteroalkyl, —(C₃-C₅)cycloalkyl,—(C₄-C₁₀)heterocyclyl, aryl, and —(C₅-C₁₀)heteroaryl, wherein the alkyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group isoptionally substituted;

wherein each occurrence of m is independently an integer ranging from 1to 5;

wherein k is an integer ranging from 1 to 5;

wherein the compound of formula (I) has a net negative charge atphysiological pH.

In another aspect, the invention provides a compound of formula (II), asalt, solvate, or N-oxide thereof:

wherein each occurrence of R¹ is independently selected from the groupconsisting of —OH, —O(C₁-C₆)alkyl, —O(C₁-C₆)haloalkyl,—O(C₁-C₆)heteroalkyl, —O(CH₂)_(m)C(═O)OR⁵, —OC(═O)R⁵, —NH₂, —SH, —SO₃Hand —PO(OH)₂;

wherein (i) R² is -X1-LINKER-CARGO or (ii) R³ is -X2-LINKER-CARGO;

-   -   X1 is a bond, —NH—, —O— or —S—;    -   X2 is —C(═O)— or —C(═S)—;    -   LINKER comprises a group consisting of —(CH₂)_(n)—,        —(CH₂CH₂O)_(n)— and -(AA)_(n)-, wherein each occurrence of n is        independently an integer ranging from 1 to 10 and wherein each        occurrence of AA is independently an amino acid, further wherein        -X-LINKER optionally further comprises a disulfide bond; and    -   CARGO is a molecule selected from an oligonucleotide,        oligodeoxynucleotide, small molecule and polypeptide;

wherein if R² is -X1-LINKER-CARGO, then R³ and R⁴ are independentlyselected from the group consisting of H, —(C═O)₀₋₁(C₁-C₆)alkyl,—(C═O)₀₋₁(C₃-C₅)cycloalkyl, —(C═O)₀₋₁(C₁-C₆)heteroalkyl, —(C═O)₀₋₁aryl,and —(C═O)₀₋₁heteroaryl, wherein the alkyl, cycloalkyl, aryl orheteroaryl group is optionally substituted; or R³ and R⁴, together withthe nitrogen to which R³ and R⁴ are connected, form—(C₃-C₁₀)heterocyclyl or —NO₂; and

wherein if R³ is -X2-LINKER-CARGO, then R² is selected from the groupconsisting of —(C₁-C₆)alkyl, —(C₁-C₆)heteroalkyl, —OR⁵,—(C₃-C₁₀)heterocyclyl, aryl, and heteroaryl, wherein the alkyl,heteroalkyl, heterocyclyl, aryl or heteroaryl group is optionallysubstituted; and R⁴ is H;

wherein each occurrence of R⁵ is independently selected from the groupconsisting of H, —(C₁-C₆)alkyl, —(C₁-C₆)heteroalkyl, —(C₃-C₅)cycloalkyl,—(C₄-C₁₀)heterocyclyl, aryl, and —(C₅-C₁₀)heteroaryl, wherein the alkyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group isoptionally substituted;

wherein each occurrence of m is independently an integer ranging from 1to 5;

wherein k is an integer ranging from 1 to 5; and

wherein the compound of formula (II) in the absence of CARGO has a netnegative charge at physiological pH.

In certain embodiments, the compound of formula (I) has a net negativecharge of −1 at physiological pH. In other embodiments, the compound offormula (I) has a net negative charge of −2 at physiological pH. In yetother embodiments, the compound of formula (I) has a net negative chargeof −3 at physiological pH. In yet other embodiments, the compound offormula (I) has a net negative charge of −4 at physiological pH. In yetother embodiments, the compound of formula (I) has a net negative chargeof −5 at physiological pH. In yet other embodiments, the compound offormula (I) has a net negative charge of −6 at physiological pH.

In certain embodiments, the compound of formula (II) in the absence ofCARGO has a net negative charge of −1 at physiological pH. In otherembodiments, the compound of formula (II) in the absence of CARGO has anet negative charge of −2 at physiological pH. In yet other embodiments,the compound of formula (II) in the absence of CARGO has a net negativecharge of −3 at physiological pH. In yet other embodiments, the compoundof formula (II) in the absence of CARGO has a net negative charge of −4at physiological pH. In yet other embodiments, the compound of formula(II) in the absence of CARGO has a net negative charge of −5 atphysiological pH. In yet other embodiments, the compound of formula (II)in the absence of CARGO has a net negative charge of −6 at physiologicalpH.

In certain embodiments, CARGO comprises a detectable label. In otherembodiments, the detectable label is selected from the group consistingof a radioisotope, stable isotope, fluorophore, electron dense metals,biotin, DNA, RNA, antibody epitope, spin label, reactive peptide tag(such as FLASH tag), quantum dot, and any combinations thereof.

In certain embodiments, at least one occurrence of R¹ is selected fromthe group consisting of —O(CH₂)_(m)C(═O)OH, —SO₃H and —PO(OH)₂. In otherembodiments, at least two occurrences of R¹ are independently selectedfrom the group consisting of —O(CH₂)_(m)C(═O)OH, —SO₃H and —PO(OH)₂.

In certain embodiments, at least one occurrence of R¹ is—O(CH₂)_(m)COOH. In other embodiments, at least two occurrences of R¹are independently —O(CH₂)_(m)COOH.

In certain embodiments, every other quinoline group in (I) has a R¹selected from the group consisting of —O(CH₂)_(m)C(═O)OH, —SO₃H and—PO(OH)₂ at the 4-position of the respective ring. In other embodiments,if a given quinoline group in (I) has a R¹ selected from the groupconsisting of —O(CH₂)_(m)C(═O)OH, —SO₃H and —PO(OH)₂ at the 4-positionof the ring, the quinoline groups to which the given quinoline group iscovalently linked have R¹ substituents that are independently selectedfrom the group consisting of —OH, —O(C₁-C₆)alkyl, —O(C₁-C₆)haloalkyl,—O(C₁-C₆)heteroalkyl, —OC(═O)R⁵, —NH₂, and —SH, at the 4-position of thecorresponding rings, wherein R⁵ is not H.

In certain embodiments, k is 1. In other embodiments, k is 2. In yetother embodiments, k is 3. In yet other embodiments, k is 4. In yetother embodiments, k is 5.

In certain embodiments, each occurrence of R¹ is independently selectedfrom the group consisting of —OCH₂CH₃ and —OCH₂COOH; NR³R⁴ is —NO₂; R²is —OMe; and k=3.

In certain embodiments each occurrence of R¹ is independently selectedfrom the group consisting of —OCH₂CH₃ and —OCH₂COOH, wherein any twoquinoline groups that are covalently linked do not have the same R¹substituent (such as, but not limited to, compounds (III) and (IV)).

In yet other embodiments, —NR³R⁴ is —NO₂ and R² is —OMe.

Exemplary compounds of the invention include formulas (III)-(IV). Thecompound of formula (III) is also denoted as ADM-116. The compound offormula (IV) is also denoted as ADM-116▮.

In certain embodiments, the compounds of the invention are watersoluble. In other embodiment, the compounds of the invention are capableof passively penetrating a cell membrane. In yet other embodiments, thecompounds of the invention reversibly folds into a cell-permeablestructure which has a hydrophobic core.

In certain embodiments, the compound of formula (II) has higher cellmembrane permeability than CARGO itself. In other embodiments, thecompound of formula (II) is cleaved within the cell to release CARGOand/or a derivative of CARGO, which has essentially the same biologicalactivity as CARGO itself.

Synthesis

The compounds of the invention may be prepared by the general schemesdescribed herein, using the synthetic method known by those skilled inthe art.

The compounds of the invention may possess one or more stereocenters,and each stereocenter may exist independently in either the (R) or (S)configuration. In certain embodiments, compounds described herein arepresent in optically active or racemic forms. In other embodiments,compounds described herein comprise a covalently linked chiral auxiliary(such as but not limited to a camphenyl group). In yet otherembodiments, compounds described herein interact with a target molecule,and this interaction inducing the folding of the compounds into a targetdriven biased folded state. It is to be understood that the compoundsdescribed herein encompass racemic, optically-active, regioisomeric andstereoisomeric forms, or combinations thereof that possess thetherapeutically useful properties described herein. Preparation ofoptically active forms is achieved in any suitable manner, including byway of non-limiting example, by resolution of the racemic form withrecrystallization techniques, synthesis from optically-active startingmaterials, chiral synthesis, or chromatographic separation using achiral stationary phase. In certain embodiments, a mixture of one ormore isomer is utilized as the therapeutic compound described herein. Inother embodiments, compounds described herein contain one or more chiralcenters. These compounds are prepared by any means, includingstereoselective synthesis, enantioselective synthesis and/or separationof a mixture of enantiomers and/or diastereomers. Resolution ofcompounds and isomers thereof is achieved by any means including, by wayof non-limiting example, chemical processes, enzymatic processes,fractional crystallization, distillation, and chromatography.

The methods and formulations described herein include the use ofN-oxides (if appropriate), crystalline forms (also known as polymorphs),solvates, amorphous phases, and/or pharmaceutically acceptable salts ofcompounds having the structure of any compound of the invention, as wellas metabolites and active metabolites of these compounds having the sametype of activity. Solvates include water, ether (e.g., tetrahydrofuran,methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetatesand the like. In certain embodiments, the compounds described hereinexist in solvated forms with pharmaceutically acceptable solvents suchas water, and ethanol. In other embodiments, the compounds describedherein exist in unsolvated form.

In certain embodiments, the compounds of the invention may exist astautomers. All tautomers are included within the scope of the compoundspresented herein.

In certain embodiments, compounds described herein are prepared asprodrugs. A “prodrug” refers to an agent that is converted into theparent drug in vivo. In certain embodiments, upon in vivoadministration, a prodrug is chemically converted to the biologically,pharmaceutically or therapeutically active form of the compound. Inother embodiments, a prodrug is enzymatically metabolized by one or moresteps or processes to the biologically, pharmaceutically ortherapeutically active form of the compound.

In certain embodiments, sites on, for example, the aromatic ring portionof compounds of the invention are susceptible to various metabolicreactions. Incorporation of appropriate substituents on the aromaticring structures may reduce, minimize or eliminate this metabolicpathway. In certain embodiments, the appropriate substituent to decreaseor eliminate the susceptibility of the aromatic ring to metabolicreactions is, by way of example only, a deuterium, a halogen, or analkyl group.

Compounds described herein also include isotopically-labeled compoundswherein one or more atoms is replaced by an atom having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number usually found in nature. Examples of isotopes suitablefor inclusion in the compounds described herein include and are notlimited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ¹³N, ¹⁵N, ¹⁵O,¹⁷O, ¹⁸O, ³²P, and ³⁵S. In certain embodiments, isotopically-labeledcompounds are useful in drug and/or substrate tissue distributionstudies. In other embodiments, substitution with heavier isotopes suchas deuterium affords greater metabolic stability (for example, increasedin vivo half-life or reduced dosage requirements). In yet otherembodiments, substitution with positron emitting isotopes, such as ¹¹C,¹⁸F, ¹⁵O and ¹³N, is useful in Positron Emission Topography (PET)studies for examining substrate receptor occupancy. Isotopically-labeledcompounds are prepared by any suitable method or by processes using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed.

In certain embodiments, the compounds described herein are labeled byother means, including, but not limited to, the use of chromophores orfluorescent moieties, bioluminescent labels, or chemiluminescent labels.

The compounds described herein, and other related compounds havingdifferent substituents are synthesized using techniques and materialsdescribed herein and as described, for example, in Fieser & Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive OrganicTransformations (VCH Publishers Inc., 1989), March, Advanced OrganicChemistry 4^(th) Ed., (Wiley 1992); Carey & Sundberg, Advanced OrganicChemistry 4th Ed., Vols. A and B (Plenum 2000,2001), and Green & Wuts,Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all ofwhich are incorporated by reference for such disclosure). Generalmethods for the preparation of compound as described herein are modifiedby the use of appropriate reagents and conditions, for the introductionof the various moieties found in the formula as provided herein.

Compounds described herein are synthesized using any suitable proceduresstarting from compounds that are available from commercial sources, orare prepared using procedures described herein.

In certain embodiments, reactive functional groups, such as hydroxyl,amino, imino, thio or carboxy groups, are protected in order to avoidtheir unwanted participation in reactions. Protecting groups are used toblock some or all of the reactive moieties and prevent such groups fromparticipating in chemical reactions until the protective group isremoved. In other embodiments, each protective group is removable by adifferent means. Protective groups that are cleaved under totallydisparate reaction conditions fulfill the requirement of differentialremoval.

In certain embodiments, protective groups are removed by acid, base,reducing conditions (such as, for example, hydrogenolysis), and/oroxidative conditions. Groups such as trityl, dimethoxytrityl, acetal andt-butyldimethylsilyl are acid labile and are used to protect carboxy andhydroxy reactive moieties in the presence of amino groups protected withCbz groups, which are removable by hydrogenolysis, and Fmoc groups,which are base labile. Carboxylic acid and hydroxy reactive moieties areblocked with base labile groups such as, but not limited to, methyl,ethyl, and acetyl, in the presence of amines that are blocked with acidlabile groups, such as t-butyl carbamate, or with carbamates that areboth acid and base stable but hydrolytically removable.

In certain embodiments, carboxylic acid and hydroxy reactive moietiesare blocked with hydrolytically removable protective groups such as thebenzyl group, while amine groups capable of hydrogen bonding with acidsare blocked with base labile groups such as Fmoc. Carboxylic acidreactive moieties are protected by conversion to simple ester compoundsas exemplified herein, which include conversion to alkyl esters, or areblocked with oxidatively-removable protective groups such as2,4-dimethoxybenzyl, while co-existing amino groups are blocked withfluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and are subsequentlyremoved by metal or pi-acid catalysts. For example, an allyl-blockedcarboxylic acid is deprotected with a palladium-catalyzed reaction inthe presence of acid labile t-butyl carbamate or base-labile acetateamine protecting groups. Yet another form of protecting group is a resinto which a compound or intermediate is attached. As long as the residueis attached to the resin, that functional group is blocked and does notreact. Once released from the resin, the functional group is availableto react.

Typically blocking/protecting groups may be selected from:

Other protecting groups, plus a detailed description of techniquesapplicable to the creation of protecting groups and their removal aredescribed in Greene & Wuts, Protective Groups in Organic Synthesis, 3rdEd., John Wiley & Sons, New York, N.Y., 1999, and Kocienski, ProtectiveGroups, Thieme Verlag, New York, N.Y., 1994, which are incorporatedherein by reference for such disclosure.

Compositions

The invention includes a pharmaceutical composition comprising at leastone compound of the invention. In certain embodiments, the compositionfurther comprises at least one additional therapeutic agent that treatsor prevents diabetes.

In certain embodiments, the compound or pharmaceutical composition andthe at least one additional therapeutic agent are co-administered to thesubject. In other embodiments, the compound or pharmaceuticalcomposition and the at least one additional therapeutic agent areco-formulated.

Methods

The invention includes a method of treating or preventing diabetes in asubject in need thereof. The invention further includes a method oftreating or preventing a neurodegenerative disease based on misfoldedand/or unstructured proteins, such Parkinson's and/or Alzheimer'sDisease.

The method comprises administering to the subject a therapeuticallyeffective amount of at least one compound of the invention, which isoptionally in a pharmaceutical composition. In certain embodiments, themethod further comprises administering to the subject an additionaltherapeutic agent that treats or prevents diabetes.

In certain embodiments, the diabetes is type I and/or type II diabetes.In other embodiments, the subject is a mammal. In yet other embodiments,the mammal is a human.

The method further includes a method of increasing the cell membranepermeability of a molecule, wherein the molecule is selected from thegroup consisting of an oligonucleotide, oligodeoxynucleotide, smallmolecule (defined as having <2,000 amu) and polypeptide, the methodcomprising derivatizing the molecule to form a compound of formula (II).In certain embodiments, the compound of formula (II) is cleaved withinthe cell releasing the molecule. In other embodiments, the compound offormula (II) is cleaved within the cell releasing a derivative of themolecule that has essentially the same biological activity as themolecule itself.

Combination Therapies

The compounds useful within the methods of the invention may be used incombination with one or more additional therapeutic agents useful fortreating diabetes. These additional therapeutic agents may comprisecompounds that are commercially available or synthetically accessible tothose skilled in the art. These additional therapeutic agents are knownto treat, prevent, or reduce the symptoms of diabetes.

In non-limiting examples, the compounds useful within the invention maybe used in combination with at least one kind of an agent selected fromthe group consisting of insulin preparations, insulin derivatives,insulin-like agonists, insulin secretagogues, insulin sensitizers,biguanides, gluconeogenesis inhibitors, sugar absorption inhibitors,renal glucose re-uptake inhibitors, β3 adrenergic receptor agonists,glucagon-like peptide-1, analogues of glucagon-like peptide-1,glucagon-like peptide-1 receptor agonists, dipeptidyl peptidase IVinhibitors, glycogen synthase kinase-3 inhibitors, glycogenphosphorylase inhibitors, anorexic agents and lipase inhibitors. It isfurther possible to combine them with at least one kind of an agentselected from the group consisting of insulin preparations, insulinderivatives, insulin-like agonists, insulin secretagogues, insulinsensitizers, biguanides, gluconeogenesis inhibitors, sugar absorptioninhibitors, renal glucose re-uptake inhibitors, β3 adrenergic receptoragonists, glucagon-like peptide-1, analogues of glucagon-like peptide-1,glucagon-like peptide-1 receptor agonists, dipeptidyl peptidase IVinhibitors, glycogen synthase kinase-3 inhibitors and glycogenphosphorylase inhibitors; and it is possible to combine them with atleast one kind of an agent selected from the group consisting of insulinpreparations, insulin derivatives, insulin-like agonists, insulinsecretagogues, insulin sensitizers, biguanides, gluconeogenesisinhibitors, sugar absorption inhibitors and renal glucose re-uptakeinhibitors. Among these, particular non-limiting examples are insulin;gliclazide, glimepiride and glibenclamide which are sulfonylurea agents;nateglinide, repaglinide and mitiglinide which are meglitinides;pioglitazone and rosiglitazone which are glitazones; metformin,phenformin and buformin which are biguanides; and acarbose, vogliboseand miglitol which are α-glucosidase inhibitors.

A synergistic effect may be calculated, for example, using suitablemethods such as, for example, the Sigmoid-E_(max) equation (Holford &Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol.114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22:27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the subjecteither prior to or after the onset of a disease or disorder. Further,several divided dosages, as well as staggered dosages may beadministered daily or sequentially, or the dose may be continuouslyinfused, or may be a bolus injection. Further, the dosages of thetherapeutic formulations may be proportionally increased or decreased asindicated by the exigencies of the therapeutic or prophylacticsituation.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat a disease or disorder in the patient. An effective amount ofthe therapeutic compound necessary to achieve a therapeutic effect mayvary according to factors such as the state of the disease or disorderin the patient; the age, sex, and weight of the patient; and the abilityof the therapeutic compound to treat a disease or disorder in thepatient. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. A non-limitingexample of an effective dose range for a therapeutic compound of theinvention is from about 1 and 5,000 mg/kg of body weight/per day. One ofordinary skill in the art would be able to study the relevant factorsand make the determination regarding the effective amount of thetherapeutic compound without undue experimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of a disease or disorder in a patient.

In certain embodiments, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Incertain embodiments, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,sodium chloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions may bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin.

In certain embodiments, the compositions of the invention areadministered to the patient in dosages that range from one to five timesper day or more. In other embodiments, the compositions of the inventionare administered to the patient in range of dosages that include, butare not limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It is readily apparent to oneskilled in the art that the frequency of administration of the variouscombination compositions of the invention varies from individual toindividual depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regime and the precise dosage and composition tobe administered to any patient is determined by the attending physicaltaking all other factors about the patient into account.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg toabout 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80mg to about 500 mg, and any and all whole or partial incrementstherebetween.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound as described herein isless than about 1,000 mg, or less than about 800 mg, or less than about600 mg, or less than about 500 mg, or less than about 400 mg, or lessthan about 300 mg, or less than about 200 mg, or less than about 100 mg,or less than about 50 mg, or less than about 40 mg, or less than about30 mg, or less than about 25 mg, or less than about 20 mg, or less thanabout 15 mg, or less than about 10 mg, or less than about 5 mg, or lessthan about 2 mg, or less than about 1 mg, or less than about 0.5 mg, andany and all whole or partial increments thereof.

In certain embodiments, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the invention, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof a disease or disorder in a patient.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical. The compounds for use in the invention may beformulated for administration by any suitable route, such as for oral orparenteral, for example, transdermal, transmucosal (e.g., sublingual,lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (intra)nasal and (trans)rectal), intravesical,intrapulmonary, intraduodenal, intragastrical, intrathecal,subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Thecompositions intended for oral use may be prepared according to anymethod known in the art and such compositions may contain one or moreagents selected from the group consisting of inert, non-toxicpharmaceutically excipients that are suitable for the manufacture oftablets. Such excipients include, for example an inert diluent such aslactose; granulating and disintegrating agents such as cornstarch;binding agents such as starch; and lubricating agents such as magnesiumstearate. The tablets may be uncoated or they may be coated by knowntechniques for elegance or to delay the release of the activeingredients. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertdiluent.

For oral administration, the compounds of the invention may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,polyvinylpyrrolidone, hydroxypropylcellulose orhydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose,microcrystalline cellulose or calcium phosphate); lubricants (e.g.,magnesium stearate, talc, or silica); disintegrates (e.g., sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Ifdesired, the tablets may be coated using suitable methods and coatingmaterials such as OPADRY™ film coating systems available from Colorcon,West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-PType, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White,32K18400). Liquid preparation for oral administration may be in the formof solutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or melting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds of theinvention, and a further layer providing for the immediate release of amedication for treatment of G-protein receptor-related diseases ordisorders. Using a wax/pH-sensitive polymer mix, a gastric insolublecomposition may be obtained in which the active ingredient is entrapped,ensuring its delayed release.

Parenteral Administration

For parenteral administration, the compounds of the invention may beformulated for injection or infusion, for example, intravenous,intramuscular or subcutaneous injection or infusion, or foradministration in a bolus dose and/or continuous infusion. Suspensions,solutions or emulsions in an oily or aqueous vehicle, optionallycontaining other formulatory agents such as suspending, stabilizingand/or dispersing agents may be used.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;5,582,837; and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and20020051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041; WO03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In certain embodiments, the formulations of the present invention maybe, but are not limited to, short-term, rapid-offset, as well ascontrolled, for example, sustained release, delayed release andpulsatile release formulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release which is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material which provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the invention may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In one embodiment of the invention, the compounds of the invention areadministered to a patient, alone or in combination with anotherpharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound of thepresent invention depends on the age, sex and weight of the patient, thecurrent medical condition of the patient and the progression of adisease or disorder in the patient being treated. The skilled artisan isable to determine appropriate dosages depending on these and otherfactors.

A suitable dose of a compound of the present invention may be in therange of from about 0.01 mg to about 5,000 mg per day, such as fromabout 0.1 mg to about 1,000 mg, for example, from about 1 mg to about500 mg, such as about 5 mg to about 250 mg per day. The dose may beadministered in a single dosage or in multiple dosages, for example from1 to 4 or more times per day. When multiple dosages are used, the amountof each dosage may be the same or different. For example, a dose of 1 mgper day may be administered as two 0.5 mg doses, with about a 12-hourinterval between doses.

It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the inhibitor of the invention isoptionally given continuously; alternatively, the dose of drug beingadministered is temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug holiday”). The length of the drugholiday optionally varies between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days,120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,320 days, 350 days, or 365 days. The dose reduction during a drugholiday includes from 10%-100%, including, by way of example only, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, is reduced to a level at which theimproved disease is retained. In certain embodiments, patients requireintermittent treatment on a long-term basis upon any recurrence ofsymptoms and/or infection.

The compounds for use in the method of the invention may be formulatedin unit dosage form. The term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosage for patients undergoingtreatment, with each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect,optionally in association with a suitable pharmaceutical carrier. Theunit dosage form may be for a single daily dose or one of multiple dailydoses (e.g., about 1 to 4 or more times per day). When multiple dailydoses are used, the unit dosage form may be the same or different foreach dose.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD₅₀ and ED₅₀. The data obtained from cell cultureassays and animal studies are optionally used in formulating a range ofdosage for use in human. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withminimal toxicity. The dosage optionally varies within this rangedepending upon the dosage form employed and the route of administrationutilized.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseExamples, but rather should be construed to encompass any and allvariations which become evident as a result of the teaching providedherein.

Methods and Materials Materials

Thioflavin T (ThT) was purchased from Acros Organics (Fair Lawn, N.J.),lipids [dioleoylphosphatidylglycerol (DOPG) anddioleoylphosphatidylcholine (DOPC)] from Avanti Polar Lipids, Inc.(Alabaster, Ala.), 96-well plates (black, w/flat bottom) from GreinerBio-One (Monroe, N.C., USA), silica plates (w/UV254, aluminium backed,200 micron) and silica gel (standard grade, particle size 40-63 micron,230×400 mesh) from Sorbent Technologies (Atlanta, Ga., USA), drysolvents from Sigma Aldrich (St. Louis, Mich.) or VWR (Bridgeport, N.J.,USA), 2,6-dichloro-3-nitropyridine, alkyl iodides, triethylamine (dry),2-chloro-1-methylpyridinium iodide, tert-butyl bromoacetate,trifluoroacetic acid (TFA), and triethylsilane (TES) from Sigma Aldrich(St. Louis, Mich., USA) and islet amyloid polypeptide (IAPP) fromGenscript (Piscataway, N.J., USA) and Elim Biopharmaceuticals (Hayward,Calif., USA). IAPP was repurified and handled in-house as follows: IAPP(˜2 mg) was solubilized in 7 M guanidinium hydrochloride. The solutionwas filtered (0.2 micron) and transferred to C-18 spin column, washedtwice with water (400 μL each) followed by 10% acetonitrile in water,0.1% formic acid (v/v) and then eluted into 200 μL of 50% acetonitrilein water, 0.1% formic acid (v/v). The concentration of IAPP wascalculated at 280 nm (ε=1400 M⁻¹·cm⁻¹). The IAPP solution was dividedinto several aliquots (20-50 μL, 1-2 mM), lyophilized, and stored as awhite solid at −80° C. A fresh stock solution of IAPP was prepared inwater from lyophilized aliquots for each experiment. Alexa-594 labelledIAPP was prepared (Magzoub, et al., FASEB J. 2012, 26, 1228-1238).

Unilameller Vesicles

Unless otherwise stated, LUVs used in this work were prepared from a 1:1mixture of DOPG/DOPC. The lyophilized mixture was hydrated in 100 mMKCl, 50 mM sodium phosphate, and pH 7.4 for 20 minutes. A 20 mg/mLsolution of lipid in buffer was passed 21 times through a polycarbonatemembrane (pore diameter=100 nm). The phospholipid content of the finalmaterial was measured using a total phosphorous assay (Chen, et al.,Anal. Chem. 1956, 28, 1756-1758).

Kinetic Assay

Amyloid reactions were conducted in buffer containing liposome (630 μMlipid) and 20 μM ThT in a black 96-well plate. This was followed byaddition of small molecule dissolved in DMSO (final DMSOconcentration=0.5%, v/v). Fibre formation was initiated by addition ofIAPP stock solution. The final volume in each well was 200 μL. Kineticsof fibrillation was monitored by ThT fluorescence (Ex 450 nm and Em 485nm) using a FluoDia T70 fluorescence plate reader (Photon TechnologyInternational, Edison, N.J., USA). The data were blank subtracted andrenormalized to the maximum intensity of reactions containing only IAPP.Each kinetic trace was fit to a sigmoidal form:

$\begin{matrix}{I = \frac{( {b_{2} + {m_{2}t}} ) + {( {b_{1} + {m_{1}t}} )e^{\frac{{t50} - t}{\tau}}}}{1 + e^{\frac{{t50} - t}{\tau}}}} & (1)\end{matrix}$

where I is the fluorescence intensity, t is time, and b, m, and r aredependent fitting variables. All samples were run at least in triplicateand error bars shown in the text represent ±one standard deviation.

Transmission Electron Microscopy

10 μM IAPP was incubated in buffer (100 mM KCl, 50 mM sodium phosphate,pH 7.4) with 630 μM LUVs in the presence and absence of IAPP:smallmolecule=1:1. Aliquots were incubated for 60 s on glow-discharged (25 mAand 30 s) carbon-coated 300-mesh copper grids. After drying, grids werenegatively stained for 60 s with uranyl acetate (2%, w/v). Micrographswere taken on a Phillips Tecnai 12 transmission electron microscope(Hillsboro, Oreg., USA) at 120 kV accelerating voltages. All conclusionsdrawn from images in this work include at least one repeat in which thesample identity was withheld from the investigator preparing andanalysing images.

Isothermal Calorimetry Titration (ITC)

ITC experiments were conducted in a NANO-ITC (TA instruments, NewCastle, Del., USA). Solutions of small molecules (100 μM in 1 mM KCl, 20mM Tris, pH 7.4) was serially titrated (50 μL injections via rotarysyringe) into an isothermal sample cell containing 10 μM IAPP (250 μL in1 mM KCl, 20 mM Tris, pH 7.4) with a stirring speed of 300 rpm. 10 sinjections were spread 240 s apart. The heat associated with eachinjection (oligoquinoline: IAPP) was extracted by integrating area undereach curve using NanoAnalyze software (New Castle, Del., USA). The heatof small molecule binding to IAPP was corrected by subtracting heat ofoligoquinoline injections into buffer. The final corrected heats wereplotted as a function of molar ratio (oligoquinoline:IAPP) and fittedwith a one binding site model.

Circular Dichroism

CD spectra were collected on AVIV MODEL 215 (AVIV Instruments, Inc.Lakewood, N.J., USA) for 25 μM IAPP mixed with 1200 μM LUVs in 100 mMKCl and 50 mM sodium phosphate at pH 7.4. The data was collected from200 to 260 nm at 0.5 nm intervals with 10 s averaging time and anaverage of 4 repeats. The CD spectra in the presence of oligoquinolineswere recorded as above except collecting from 200 to 500 nm at astoichiometric ratio of 1:1 (oligoquinoline:IAPP).

Small Molecule Characterization

Final steps of small molecule purification was conducted by HPLC using aVarian ProStar with VYDAC reverse-phase columns (4.6×100 mm, 1 mL/min,analytical; 10×100 mm, 3 mL/min, semiprep.). The mobile phase wascomposed of A: 5% ACN, 95% H₂O, 0.1% TFA(v/v) and B: 95% ACN, 5% H₂O,and 0.08% TFA (v/v). The solution NMR spectra of small molecules wererecorded on 400, 500, and 600 MHz Agilent spectrometers. The deuteratedsolvents used for O-tert butyl ester protected and deprotected (O—COOH)oligoquinolines were CDCl₃ and (CD₃)₂SO respectively. Splitting patternsthat were difficult to interpret, are indicated as multiplet (m) orbroad (b). Mass spectra were obtained using either MALDI-TOF Voyager DEPro (Yale University, CBIC center) or University of Illinois Mass Spec.Facility. High-resolution electrospray ionization mass spectra wereobtained using the Waters Synapt G2-Si ESI MS mass spectrometer(Milford, Mass., USA).

Fluorescence Correlation Spectroscopy

FCS measurements were made on a laboratory-built instrument based aroundan inverted microscope using an Olympus IX71 microscope (Olmpus, Tokyo,Japan), as described previously in Middleton, et al., Effects ofCurvature and Composition on α-Synuclein Binding to Lipid Vesicles(Biophys. J. 2010, 99, 2279-2288). Briefly, a continuous-emission 488-nmdiode-pumped solid-state 50 mW laser was set to 5-20 mW output power andfurther adjusted with neutral density filters to 18 μW of power justprior to entering the microscope. Fluorescence was collected through theobjective and separated from the excitation laser using a Z488rdclong-pass dichroic and an HQ600/200m bandpass filter (Chroma, BellowsFalls, Vt., USA). Fluorescence was focused onto the aperture of a 50 μmoptical fibre coupled to an avalanche photodiode (Perkin Elmer, Waltham,Mass., USA). A digital correlator (Flex03LQ-12; Correlator.com,Bridgewater, N.J., USA) was used to generate autocorrelation curves.

Measurements were made in 8-well chambered coverglasses (Nunc,Rochester, N.Y., USA) which were plasma treated followed by precoatingwith polylysine-conjugated polyethylene glycol (PEG-PLL), to preventADM-116 and/or IAPP from adsorbing to chamber surfaces. Low density PEGcoating was preformed by preparing a 100 mg/ml solution of PEG (MW=2KDa, NANOCS, Boston, Mass., USA) in Poly-L-Lysine hydrobromide (SigmaAldrich, St. Louis, Mich., USA). Reaction was preformed for 6 h in darkat room temperature, followed by overnight dialysis. Chambers wereincubated overnight with PEG-PLL solution, rinsed thoroughly withMillipore water, and stored in water before use. All samples wereincubated in buffer (20 mM Tris, pH 7.4, 10 mM NaCl) for 1 h prior totaking measurements. Titration was performed keeping constantconcentrations of ADM-116_(F) (25 nM) and adding increasingconcentrations of IAPP. The autocorrelation curves were collected atregular intervals (10 min), and each autocorrelation curve was collectedover 10 sand repeated 30 times.

Autocorrelation curves were fitting using Matlab (The MathWorks,Nattick, Mass., USA) and further global analysis were performed onIgorPro (ADInstruments, Colorado Springs, Colo., USA). For ADM-116, themodel for a single diffusing species undergoing 3D Brownian diffusionwith a triplet state is given by Eq. 2.

$\begin{matrix}{{G(\tau)} = {\frac{1}{N( {1 - T} )} \times ( {1 - T + {Te}^{({{- t}/\tau_{triplet}})}} ) \times \lbrack {1 + \frac{\tau}{\tau_{d,1}}} \rbrack^{- 1} \times \lbrack {1 + \frac{\tau}{s^{2}\tau_{d,1}}} \rbrack^{- \frac{1}{2}}}} & (2)\end{matrix}$

Here, Nis the number of ADM-116_(F) molecules in the detection volume,Tis the fraction of molecules in the triplet state and τ_(triplet) isthe triplet state relaxation time. The characteristic translationaldiffusion time of a diffusing particle is given by τ_(d,1).

In the presence of IAPP, the model for two-component analysis is givenby:

$\begin{matrix}{{{{G(\tau)} = {\frac{1}{N( {1 - T} )} \times ( {1 - T + {Te}^{({{- t}/\tau_{triplet}})}} ) \times}}\quad}{\quad\lbrack {{r \times \lbrack {1 + \frac{\tau}{\tau_{d,1}}} \rbrack^{- 1} \times \lbrack {1 + \frac{\tau}{s^{2}\tau_{d,1}}} \rbrack^{- \frac{1}{2}}} + {( {1 - r} ) \times \lbrack {1 + \frac{\tau}{\tau_{d,2}}} \rbrack^{- 1} \times \lbrack {1 + \frac{\tau}{s^{2}\tau_{d,2}}} \rbrack^{- \frac{1}{2}}}} \rbrack}} & (3)\end{matrix}$

where r is the fraction of the fast-diffusing component with a diffusiontime τ_(d,1), whereas τ_(d,2) is the diffusion time of the slowcomponent. The structure factor, s, was determined as a free parameterfor solutions of free Alexa Fluor 488 hydrazide dye and then fixed tothe experimentally determined value of 0.17 for all subsequent fittings.For experiments in the presence of IAPP, global analysis was performedby fixing the predetermined values for the diffusion coefficient,triplet diffusion time, and amplitude for ADM-116. The triplet state ofIAPP bound and unbound ADM-116 were considered to be the same.

Confocal Microscopy

Images were obtained in 8 well NUNC chambers (Thermo Scientific,Rochester, N.Y., USA) seeded with 20000-25000 cells/well. Afterculturing for 48 h, the medium was replaced with medium containingconstituents according to the experiment performed. For time dependentlocalization experiments of IAPP, the medium contained 100 nM IAPP₅₉₄,13 μM unlabelled peptide and incubated for the specified time points.For experiments in the presence of ADM-116_(F) and ADM-3_(F), additionalfluorescein labelled and unlabelled small molecules, 200 nM and 13 μM,respectively was introduced in the medium. For delayed additionexperiment with small molecules, the medium was removed for the secondtime, replacing with medium containing the small molecule. Images wereacquired after 48 h total incubation time. Imaging was carried out atthe Yale Department of Molecular, Cellular, and Developmental Biologyimaging facility, on a Zeiss LSM 510 confocal microscope, using a x63Plan-Apo/1.4-NA oil-immersion objective with DIC capability (Carl Zeiss,Oberkochen, Germany). For all experiments reporting on the uptake oflabelled IAPP, the gain setting for the red channel was kept constantfrom sample to sample. Image acquisition and processing were achievedusing Zeiss Efficient Navigation (ZEN) and Image J software.

Imaging FRET (Forster Resonance Energy Transfer)

The INS-1 growth media was then replaced with media containing 100 nMIAPP₅₉₄ and incubated for 18 h. Media was then replaced a second timewith media containing 200 nM ADM-116_(F). Images were taken after 5 hincubation. Background FRET was determined using parallel experimentswhere 100 nM IAPP₅₉₄ was initially incubated for 18 h in cells in thepresence of a further 13 μM of unlabelled IAPP. Media was then replacedwith media containing 200 nM ADM-116_(F). Imaging was carried out on aZeiss LSM 510 confocal microscope, using a x100 Plan-Apo/1.4-NAoil-immersion objective with DIC capability (Carl Zeiss, Oberkochen,Germany). For ADM-116_(F), fluorescein was excited with a 488 nm Argon2laser and detected through a 505-550 nm emission filter. IAPP₅₉₄ wasexcited with a 561 Argon2 laser and detected through a 590-630 nmemission filter. For all experiments the pinhole was kept constant tothe Z-slick thickness of each filter channel. Single cell images wereobtain for donor alone, acceptor alone and donor-acceptor fusionchannels. Image acquisition and processing were achieved using ZeissEfficient Navigation (ZEN) and Image J software. The Image J plugin,RiFRET, was used to calculate and remove the bleed through for eachchannel and to calculate a pixel-based FRET efficiency. The FRETdistance was then calculated using:

$\begin{matrix}{E = \frac{R_{0}^{6}}{R_{0}^{6} + r^{6}}} & (4)\end{matrix}$

Where E is the calculated efficiency of FRET energy transfer, R₀ is theForster distance (60 Å for fluorescein-Alexa594 pair) and r is thedistance between the donor and the acceptor.

Cell Culture

Rat insulinoma INS-1 cells (832/13, Dr. Gary W. Cline, Department ofInternal Medicine, Yale University) were cultured at 37° C. and 5% CO₂in phenol red free RPMI 1640 media supplemented with 10% fetal bovineserum, 1% penicillin/streptomycin (Life Technologies, Carlsbad, Calif.,USA), and 2% INS-1 stock solution (500 mM HEPES, 100 mM L-glutamine, 100mM sodium pyruvate, and 2.5 mM β-mercaptoethanol). Cells were passagedupon reaching ˜95% confluence (0.25% Trypsin-EDTA, Life Technologies),propagated, and/or used in experiments. Cells used in experiments werepelleted and resuspended in fresh media with no Trypsin-EDTA.

Giant Plasma Membrane Vesicle (GPMV) Isolation

GPMVs were isolated from INS-1 cells (Schlamadinger et al., Biophys. J.2014, 107, 2559-2566, Kendhale et al., J. Org. Chem. 2011, 76, 195-200).Briefly, cells were plated in 35 mm dishes and cultured for 48 h. Cellswere washed with a 10 mM HEPES, 150 mM NaCl, 2 mM CaCl₂) (pH=7.4) twiceand were then exposed to 10 mM N-ethyl maleimide (NEM, Sigma Aldrich,St. Louis, Mich., USA) for 2 h. Collected samples were then passed overa gravity-flow column (Bio-Rad) containing size exclusion Sephacrylmatrix of pore size 400-HR (Sigma Aldrich, St. Louis, Mich., USA)allowing the purification of GPMVs from residual cell debris.

GPMV Imaging

Images were obtained in 8 well NUNC chambers (Thermo Scientific,Rochester, N.Y., USA) including 250 μl of GMPV stock solution. Forexperiments in the presence of ADM-116_(F) or ADM-3F, 200 nM of smallmolecule was incubated in the GMPV solution for 24 h at roomtemperature. Imaging was carried out at the Yale Department ofMolecular, Cellular, and Developmental Biology imaging facility, on aZeiss LSM 510 confocal microscope, using a x63 Plan-Apo/1.4-NAoil-immersion objective with DIC capability (Carl Zeiss, Oberkochen,Germany). For all experiments, the gain setting for the green channelwas kept constant from sample to sample. Image acquisition andprocessing were achieved using Zeiss Efficient Navigation (ZEN) andImage J software.

Cell Viability

Cell viability was measured colourimetrically using the Cell-Titer Blue(CTB, Promega, Madison, Wis., USA) fluorescence-based assay. Cells wereplated at a density 5000 cells/well in 96-well plates (BD Biosciences,San Diego, Calif.). Peptide was directly introduced to each well after48 h of culture and then further incubated for an additional 48 h. Fortime dependent experiments, cells were incubated with peptide for thespecified time points. After the incubation period, 20 μL CTB reagentwas added to each well and incubated at 37° C. and 5% CO₂ for 2.5-3.5 h.Fluorescence of the resorufin product was measured on a FluoDia T70fluorescence plate reader (Photon Technology International, Birmingham,N.J., USA). All wells included the same amount of water to account fordifferent concentrations of peptide added to sample wells. Wells thatincluded water vehicle but not peptide served as the negative control(0% toxic), and wells containing 10% DMSO were the positive control(100% toxic). Percent toxicity was calculated using the followingequation:

$\begin{matrix}{{\%\mspace{14mu}{Toxicity}} = {100 - \lbrack {100 \cdot ( \frac{< S > {- {< P >}}}{< N > {- {< P >}}} )} \rbrack}} & (5)\end{matrix}$

Each independent variable is the average of eight plate replicates fromthe negative control (<N>), positive control (<P>), and samples (<S>).Results presented for viability experiments are an average of three suchexperiments conducted independently on different days. Error barsrepresent the standard error of the mean.

Apoptosis was measured colourimetrically by detecting caspase 3/7(Caspase-Glo® 3/7 Assay, Promega, Madison, Wis., USA). Cells were platedat a density 5000 cells/well in 96-well plates (BD Biosciences, SanDiego, Calif., USA). Peptide was directly introduced to each well after48 h of culture and then further incubated for the times specified inthe main text. After the incubation period, 20 μL Caspase-Glo® 3/7reagent (containing a mixture of caspase-3/7 DEVD-aminoluciferinsubstrate and a proprietary thermostable luciferase in a reagentoptimized for caspase-3/7 activity) was added to each well and incubatedat 37° C. and 5% CO₂ for 2 h. Fluorescence of the free aminoluciferinproduct was measured on a FluoDia T70 fluorescence plate reader (PhotonTechnology International, Birmingham, N.J., USA). For proteinconcentration dependence measurements, carrier buffer was added asrequired to ensure identical volumes of protein were added to each well.

Example 1: Time Dependent Localization of IAPP

INS-1 cells were incubated with 100 nM IAPP labelled at its N-terminuswith Alexa-594 (IAPP_(A594)). Co-addition of 0 μM or 13 μM unlabelledIAPP corresponds to non-toxic and toxic conditions respectively. At 5 h,under non-toxic conditions, IAPP was not significantly internalized(FIG. 2). By 12 h, intracellular IAPP was readily observed with maximumextent of internalization apparent at 24 h (FIG. 2). At all time-pointsunder this non-toxic condition, IAPP appeared as diffuse puncta,possibly a consequence of energy dependent cellular uptake under theseconditions. Under toxic conditions, slightly elevated uptake of IAPP wasapparent by 5 h. By 12 h, contrasting behaviour could be clearly seenwith the toxic condition showing external and internalized puncta andlarger assemblies. By 24 h, extracellular IAPP was a small fraction ofthe total IAPP. At 48 h, large intracellular aggregates appeared andcontinued to increase in intensity through 72 h. This progressionsuggests that aggregation was mediated by the intracellular environment,consistent with work showing culture media and plasma membrane to beinhibitory to amyloid formation.

In certain embodiments, cytotoxicity is mediated by intracellular IAPP.The time dependence of IAPP-mediated toxicity was measured in parallelto imaging. Apoptosis was apparent as early as 5 h with caspase 3/7activity continuing to increase over the course of observation (FIG. 2).The fraction of cells affected by IAPP could be approximated bymonitoring total cytosolic reductase activity relative to IAPP-freecontrols. The trend clearly parallels the apoptosis. Importantly,continued increases in apoptotic activity were apparent long after the24 h time-point where little extracellular IAPP was evident (FIG. 2).Taken together, this suggests the site of toxicity is intracellular.Without wishing to be limited by any theory, small molecule modulationof IAPP requires a compound that can be internalized.

Example 2: Targeting Membrane Bound IAPP

There is a direct correlation between targeting membrane bound IAPP,inhibition of several solution-based gains-of-function, and cytotoxicrescue. Without wishing to be limited by any theory, compounds thattarget intracellular IAPP may be more likely to be found among membraneactive inhibitors of IAPP self-assembly. In solution, assembly of 10 μMIAPP into fibres occurred with a reaction midpoint, t₅₀, of 18±1.4 h(FIG. 9). The same reaction conducted in the presence of largeunilamellar vesicles (LUVs) was accelerated to a t₅₀ of 1.1±0.1 h (FIG.3A). The latter serves as a reference condition under which smallmolecules can be assessed for activity in the context of a bilayer.

ADM-116 (FIGS. 1A-1 i) is a small molecule with marked activity in LUVcatalysed fibrillation assays. At 1:1 (IAPP:ADM-116), liposome catalysedfibrillation was undetectable (a t₅₀>40 fold higher than control) withsignificant inhibition observable even at 1:0.1 (FIG. 3). Amyloidformation was not observed by electron microscopy (FIG. 10) and far-UVCD (FIG. 5D). In comparison, compounds ADM-3 (FIG. 15) and ADM-118inhibit by factors of 2.7±0.1 and 3.3±0.1 fold respectively. Othernatural product compounds such as EGCG, acid fuchsin (AF) andresveratrol, show no detectable effect (FIG. 3). In certain embodiments,ADM-116 contained functional moieties, the steric and physico-chemicalproperties of which result in exceptional inhibition of lipid catalysedamyloid assembly.

ADM-116 rescued IAPP induced toxicity. After 48 h of incubation with 13μM IAPP, INS-1 viability was decreased 78±8%. Co-addition of ADM-116 ata stoichiometric ratio of 1:1 (IAPP:ligand) wholly restored viability(FIGS. 4A-4B). The compound ADM-3, a tripyridylamide, was also active onmembrane bound IAPP and also restored cell viability (FIG. 4A) with adose dependence comparable to ADM-116 (FIG. 4B). Resveratrol, EGCG andacid fuchsin were ineffective in rescuing toxicity under sameconditions. The origin for this disparity was the requirement for IAPPand these small molecules to be preincubated for >11 h prior to addingthe complexes to cell culture. In contrast, IAPP and the small moleculewere co-introduced to cell culture. At concentrations investigatedherein, none of the molecules showed intrinsic toxicity towards INS-1cells, nor interfered with the colourimetric assay (FIG. 11).

ADM-116, but not ADM-3, rescued cells from intracellular IAPP toxicity.In light of the intracellular origins of toxicity suggested elsewhereherein (FIG. 2), cell viability was instead assessed under conditions inwhich introduction of IAPP was followed by a delay prior to the additionof small molecule. Remarkably, even after a delay of 24 h, ADM-116 wascapable of rescuing IAPP induced cytotoxicity by 47±4%. In markedcontrast, ADM-3 was ineffective in rescuing toxicity when added after 12h (FIG. 4C). As IAPP was internalized by 24 h of incubation (FIG. 2),this suggests that the mechanism of ADM-116 rescue includes penetrationof the plasma membrane.

Example 3: Molecular Specificity of Binding

The solution and cellular activities of ADM-116 were enabled byformation of a discrete complex with IAPP. Unlabelled IAPP was titratedinto a solution of 25 nM fluorescein labelled ADM-116 (ADM-116_(F)) andthe diffusion time of the small molecule in the absence and presence ofpeptide was monitored by fluorescence correlation spectroscopy (FCS).ADM-116_(F) alone shows a τ_(D) of 130±13 μs (FIG. 5a ). Upon titrationwith IAPP, a second component was evident with a diffusion time ofτ_(D)=˜400 s. The consistency of the diffusion time over the course ofthe titration indicates a discrete complex. Fitting the fractionalamplitude of bound to unbound ADM-116_(F) gave K_(d) of 240±60 nM. Theτ_(D) of fluorescently labelled IAPP alone was 230±4 μs. Assuming allcomponents are spheres, diffusion scales by mass. The stoichiometry ofthe complex under this assumption was 1:1. Isothermal titrationcalorimetry (ITC) using unlabelled IAPP and ADM-116 yielded anexothermic profile that clearly fitted a one site binding model with aK_(a)=3.1±0.6×10⁶ M⁻¹ (K_(d)=320±60 nM) (FIG. 5b and Table 1). Thus,ADM-116 formed a discrete complex with strong affinity to IAPP.

TABLE 1 Binding Affinities. ITC-derived thermodynamic parameters for thebinding of the indicated ligands with IAPP. Units of energy are kJ/mol.Presented errors are the standard deviation from experiments performedat least three times. Compound K_(a) × 10⁻⁶ n ΔH ΔS TΔS ΔG ADM-116 3.1 ±0.6 1.0 ± 0.1 −230 ± 5 750 ± 17 −220 ± 5  −36 ± 7 ADM-116| 0.7 ± 0.1 0.9± 0.1  −89 ± 8 190 ± 29 −57 ± 9 −33 ± 5 ADM-116_(P) 0.9 ± 0.1 1.0 ± 0.0 −340 ± 36 −1000 ± 120  −310 ± 36 −33 ± 4

ADM-116 stabilized the α-helical sub-domain of IAPP. The far-UV CDspectrum of membrane bound IAPP exhibited two minima near 208 and 222nm, characteristic of α-helical structure (FIG. 5D). This profiletransitioned to that of β-sheet, characteristic of amyloid, within 1 h.In contrast, IAPP remained predominantly α-helical even after 4 h whenincubated with ADM-116 (1:0.5, IAPP:ADM-116). α-helical structure withinthe membrane binding sub-domain of IAPP is mapped to segments containingmost of the first 22 residues of IAPP. Within this stretch, the sequencevariant of IAPP from rat, rIAPP, varies only at position 18 (H18R).Binding of ADM-116 by rIAPP was not detectable (FIG. 5B). An additionalfive residues differ between hIAPP and rIAPP over residues 23-29.Amyloid nucleation in full length hIAPP was mediated, in part, byresidues 20-29. The kinetic profile of amyloid formation by 200 μM ofthe 10-residue sub-peptide, IAPP₂₀₋₂₉, gave a t₅₀ of 6.6±0.3 h (FIG.12). The presence of 1:1 ADM-116 has little effect on the independentamyloid assembly of IAPP₂₀₋₂₉. Thus, ADM-116 did not functionallyinteract with human residues 20-29, nor does it detectably bind torIAPP. Combined with the observation of membrane bound α-helicalstabilization, this suggests that ADM-116 binds to the α-helicalsubdomain of IAPP.

Example 4: Specificity of Intracellular Binding

To gain mechanistic insight, ADM-116 was fluorescently labelled at itsOCH₃ end, applied to INS-cells and visualized by confocal microscopy.Fluorescein labelled ADM-116 (ADM-116_(F)), at non-rescue (200 nM) andrescuing (200 nM+15 μM ADM-116) concentrations, was able to penetratethe cell membrane (FIG. 6A). In contrast, fluorescein labelled ADM-3(ADM-3_(F)) showed no capacity to populate the cell interior (FIG. 6A).Giant plasma membrane derived vesicles (GPMVs) were then prepared fromlive INS-1 cells. ADM-116, and not ADM-3, showed a clear capacity topenetrate the GPMVs (FIG. 6B) indicating that not only does the formersmall molecule cross the membrane, it does so without assistance fromactive cellular processes.

Cellular rescue was associated with colocalization of protein and smallmolecule. INS-1 cells were treated with toxic concentrations of IAPP(100 nM IAPP₅₉₄ and 13 μM IAPP). After 20 h, rescuing concentrations ofADM-116 (200 nM ADM-116_(F) and 15 μM ADM-116) were added. In rescuedcells, IAPP colocalized with ADM-116 and fewer IAPP aggregates wereobserved (FIG. 7A). An alternative mechanism operated under conditionsin which IAPP and ADM-116 were cointroduced. Although IAPP and ADM-116could separately enter cells (FIG. 2, and FIG. 6A), added together, theycolocalized on the cell surface (FIG. 7B). A similar observation can bemade for ADM-3 (FIG. 13). This suggests that the interaction of ADM-116or ADM-3 with IAPP prohibited the entry of toxic peptide into the cells.In contrast, rescue by delayed addition of ADM-116 appeared to disrupttoxic aggregate formation by direct interaction.

Intracellular rescue was a consequence of direct interactions betweenIAPP and ADM-116. Forster resonance energy transfer (FRET) measurementswere made in live cells. Here, 200 nM ADM-116_(F) was applied to INS-1cells 20 h after 100 nM IAPP₅₉₄. Looking only to data above background,nonradiative energy transfer from ADM-116F to IAPP₅₉₄ was readilyapparent and intracellular (FIG. 7C). Importantly, statisticalassessment of the FRET revealed a Gaussian distribution stronglyconsistent with interactions not only being close (˜40 Å), but alsodiscrete and therefore specific (FIG. 7D).

Example 5: Structure, Dynamics and Function of ADM-116

ADM-116 is a folded molecule with a hydrophobic core. For FDA approveddrugs, measured and calculated octanol-water partition coefficients areclosely comparable (FIG. 6C). The predicted and experimental log P forADM-3 are −0.7 and −2.0, consistent with its practical solubility inbuffer (FIG. 6C). For ADM-116 however, there is a 7-order of magnitudedisparity. The calculated log P was 7.6 (comparable to cholesterol)while the measured log P was 0.6. This anomaly may be a consequence ofthe folded nature of ADM-116. Overall, ADM-116 is anionic (−2), watersoluble and yet capable of passive cell membrane penetration. Thissuggests a paradigm for cell penetration in which the folding/refoldingof the small molecule is central to this process.

Refolding of ADM-116 is affected by IAPP binding. Refolding permitsoligoquinolines to sample an equal mixture of right-(P) and left-(M)mirror-image helices. The CD spectrum of ADM-116 in aqueous buffercontained equal contributions from these two hands resulting in a flatline at 390 nm (FIG. 8B). In marked contrast, the CD spectra of ADM-116recorded in the presence of stoichiometric IAPP (FIG. 8A) showedpositive ellipticity. Binding clearly shifted the refolding equilibriumbetween right and left handed helical states.

IAPP binds preferentially to the (P) conformer of ADM-116. Two analogueswere synthesized in which chiral camphenyl groups were coupled to theNO₂ terminus of ADM-116 (FIG. 15). CD spectra confirm the induction ofhelical bias (FIG. 8B) with ADM-116_(P) and ADM-116_(M) defined as thederivatives that give positive and negative profiles at 390 nmrespectively. For neither compound could the ratio of right/left handhelical states be determined in absolute terms. Importantly, theintensity of ADM-116 in the presence of IAPP was within 90% that ofADM-116_(P) (FIG. 8A and FIG. 16). By ITC, the K_(a) of IAPP to ADM-116Pis 0.9±0.1×10⁶ M⁻¹; within a factor of three of ADM-116. In contrast,binding was not detectable for ADM-116_(M) (FIG. 5B). This pattern wasevident in diverse assays. For lipid catalysed fibrillation reactions,ADM-116_(P) was as potent an inhibitor as ADM-116 while ADM-116_(M)inhibited fibrillation only weakly (FIG. 8C). In cell toxicity assays,ADM-116_(P) showed ˜2.4 times greater inhibition of toxicity thanADM-116_(M) (FIG. 8C). Thus, in two solution biophysical assays and intoxic rescue, IAPP:ADM-116 interactions were stereospecific.

IAPP binding to ADM-116 included interactions with the OCH₃ end of theADM-116 helix. Enantiomeric interconversion in oligoquinolines is morerestricted with increasing length. Here, two molecules were synthesizedin which ADM-116 was lengthened by one subunit either at the NO₂(ADM-116▮), or OCH₃ end (ADM-▮116). The lateral surfaces of ADM-116▮ andADM-▮116 necessarily included the surface of ADM-116. If IAPP:ADM-116binding includes contact with the ends of the quinoline helix, thenbinding to one of the two variants should be more strongly affected. TheK_(a) of IAPP:ADM-116▮ was similar to ADM-116 (K_(a)=7±1×10⁶ M⁻¹). Thisbinding induced a random coil to α-helix transition comparable toADM-116 (FIG. 16B). The binder, ADM-116_(P), was also an NO₂ endderivative. In marked contrast, no binding is detected for ADM-▮116.This indicates that IAPP interacted with the OCH₃ end of ADM-116.

Intracellular toxic rescue by ADM-116 was dependent on oligoquinolinelength. In toxicity studies, the co-addition of ADM-116▮and IAPPresulted in diminished toxicity relative to IAPP alone (FIG. 4A).However, if ADM-116▮was added to INS-1 cells after IAPP wasinternalized, rescue was no longer observed (FIG. 4C). In other words,the addition of a fifth quinoline to ADM-116 resulted in a molecule thatretained activities comparable to ADM-3, i.e. a compound that is aneffective antagonist provided IAPP and small molecule encounter eachother in the extracellular environment.

Without wishing to be limited to any theory, differences in thebehaviour of ADM-116▮ and ADM-116 could be mapped to changes in therelative folded stability of the small molecule. The ΔΔG of IAPP bindingto ADM-116▮ versus ADM-116 was ˜3 kJ/mol (FIG. 5C and Table 1). Changesto entropic contributions (ΔTΔS) were ˜160 kJ/mol (FIG. 5C). If theIAPP:ADM-116▮ and IAPP:ADM-116 interfaces are structurally the same,this disparity must instead be mapped to differences in the unboundstate(s) of the oligoquinolines. The large entropic contributionresembles a hydrophobic effect; consistent with the expectation that thetetraquinoline, ADM-116, more readily samples unfolded states thanpentaquinoline, ADM-116▮. The fact that the tetraquinoline, ADM-116_(P),has a thermodynamic profile more similar to ADM-116 complements thisassertion. Structural assessment of the IAPP:ADM-116▮ complex by CDshowed weak ellipticity at 390 nm compared to IAPP:ADM-116 even afterdays of incubation (FIG. 8A). Thus, whereas binding affinity of IAPP toADM-116▮ is comparable to ADM-116, the capacity of the binding energy toshift the enantiomeric equilibrium is reduced. Taken together, theseobservations suggest that ADM-116 exists predominantly in its foldedstate, but readily samples partially and fully unfolded states.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1-23. (canceled)
 24. A method of preventing, ameliorating, or treatingdiabetes in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of atleast one compound of formula (I), or a salt, solvate, or N-oxidethereof, and any combinations thereof:

wherein each occurrence of R is independently selected from the groupconsisting of —OH, —O(C₁-C₆)alkyl, —O(C₁-C₆)haloalkyl,—O(C₁-C₆)heteroalkyl, —O(CH₂)_(m)C(═O)OR⁵, —OC(═O)R⁵, —NH₂, —SH, —SO₃Hand —PO(OH)₂; wherein R² is selected from the group consisting of—(C₁-C₆)alkyl, —(C₁-C₆)heteroalkyl, —OR⁵, —(C₃-C₁₀)heterocyclyl, aryland heteroaryl, wherein the alkyl, hetereoalkyl, heterocyclyl, aryl orheteroaryl group is optionally substituted: wherein R³ and R⁴ areindependently selected from the group consisting of H,—(C═O)₀₋₁(C₁-C₆)alkyl, —(C═O)₀₋₁(C₃-C₅)cycloalkyl,—(C═O)₀₋₁(C₁-C₆)heteroalkyl, —(C═O)₀₋₁aryl, and —(C═O)₀₋₁heteroaryl,wherein the alkyl, cycloalkyl, aryl or heteroaryl group is optionallysubstituted; or R³ and R⁴, together with the nitrogen to which R³ and R⁴are connected, form —(C₃-C₁₀)heterocyclyl or —NO₂: wherein eachoccurrence of R⁵ is independently selected from the group consisting ofH, —(C₁-C₆)alkyl, —(C₁-C₆)heteroalkyl, —(C₃-C₅)cycloalkyl,—(C₄-C₁₀)heterocyclyl, aryl, and —(C₅-C₁₀)heteroaryl, wherein the alkyl,heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group isoptionally substituted: wherein each occurrence of m is independently aninteger ranging from 1 to 5; wherein k is an integer ranging from 1 to5; and wherein the compound of formula (I) has a net negative charge atphysiological pH. 25-38. (canceled)
 39. The method of claim 24, whereinthe diabetes is type I or type II diabetes.
 40. The method of claim 24,wherein at least one occurrence of R¹ is selected from the groupconsisting of —O(CH₂)_(m)C(═O)OH, —SO₃H and —PO(OH)₂.
 41. The method ofclaim 40, wherein at least one occurrence of R¹ is —O(CH₂)_(m)COOH. 42.The method of claim 24, wherein at least two occurrences of R¹ areindependently selected from the group consisting of —O(CH₂)_(m)C(═O)OH,—SO₃H and —PO(OH)₂.
 43. The method of claim 42, wherein at least twooccurrences of R¹ are independently —O(CH₂)_(m)C(═O)OH.
 44. The methodof claim 24, wherein every other quinoline group in (I) has a R¹independently selected from the group consisting of —O(CH₂)_(m)C(═O)OH,—SO₃H and —PO(OH)₂ at the 4-position of the respective ring.
 45. Themethod of claim 44, wherein, if a given quinoline group in (I) has a R¹selected from the group consisting of —O(CH₂)_(m)C(═O)OH, —SO₃H and—PO(OH)₂ at the 4-position of the ring, the quinoline groups to whichthe given quinoline group is covalently linked have R¹ substituents thatare independently selected from the group consisting of —OH,—O(C₁-C₆)alkyl, —O(C₁-C₆)haloalkyl, —O(C₁-C₆)heteroalkyl, —OC(═O)R⁵,—NH₂, and —SH, at the 4-position of the corresponding rings, wherein R⁵is not H.
 46. The method of claim 24, wherein each occurrence of R¹ isindependently selected from the group consisting of —OCH₂CH₃ and—OCH₂COOH; NR³R⁴ is —NO₂; R² is —OMe; and k is
 3. 47. The method ofclaim 24, wherein any two quinoline groups that are covalently linked donot have the same R¹ substituent.
 48. The method of claim 24, whereinthe compound of formula (I) is the compound of formula (III) or (IV):


49. The method of claim 24, wherein at physiological pH the compound hasa net negative charge that is selected from the group consisting of −1,−2, −3, −4, −5, and −6.
 50. The method of claim 24, wherein k is
 1. 51.The method of claim 24, wherein k is
 2. 52. The method of claim 24,wherein k is
 3. 53. The method of claim 24, wherein k is
 4. 54. Themethod of claim 24, wherein k is
 5. 55. The method of claim 24, whereinthe compound is water soluble.
 56. The method of claim 24, wherein thecompound is capable of passively penetrating a cell membrane.
 57. Themethod of claim 24, wherein the compound is further coupled with adetectable label.
 58. The method of claim 57, wherein the detectablelabel is selected from the group consisting of a radioisotope, stableisotope, fluorophore, electron dense metals, biotin, DNA, RNA, antibodyepitope, spin label, reactive peptide tag, quantum dot, and anycombinations thereof.
 59. The method of claim 24, wherein the compoundis administered to the subject as a pharmaceutical composition furthercomprising at least one pharmaceutically acceptable carrier.
 60. Themethod of claim 24, further comprising administering to the subject atleast one additional therapeutic agent that treats, ameliorates, orprevents diabetes.
 61. The method of claim 60, wherein the compound andthe at least one additional therapeutic agent are co-administered to thesubject.
 62. The method of claim 60, wherein the compound and the atleast one additional therapeutic agent are co-formulated.
 63. The methodof claim 24, wherein the subject is a human.